KR101882523B1 - Vista regulatory t cell mediator protein, vista binding agents and use thereof - Google Patents

Abstract

The present invention relates to a novel regulatory T cell protein. This protein, termed PD-L3 or VISTA, is similar to members of the PD-L1 family and has been identified and is a novel, structurally distinct Ig-superfamily inhibitory ligand, The extracellular domain has homology with the B7-based ligand PD-L1. This molecule was named PD-L3 or a V-domain immunoglobulin inhibitor (VISTA) of VISTA or T cell activation. Expression of VISTA is usually within the hematopoietic component and is highly regulated in myeloid APCs and T cells. Therapeutic intervention of the VISTA inhibitory pathway represents a novel approach to controlling T-cell mediated immunity for the treatment of various cancers, such as ovarian cancer, bladder cancer and melanoma. In addition, VISTA proteins, particularly multimeric VISTA proteins and antibodies, can be used to inhibit T cell immunity in autoimmune diseases, allergies, infectious and inflammatory diseases, such as multiple sclerosis and arthritic diseases such as RA.

Description

VISTA REGULATORY T CELL MEDIATOR PROTEIN, VISTA BINDING AGENTS AND USE THEREOF BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

This application is a continuation-in-part of US Serial No. < RTI ID = 0.0 > No. 12 < / RTI > 732,371, filed Oct. 6, 2010; U.S. Provisional Application No. 61 / 390,434, filed January 26, 2011; 61 / 436,379 filed March 7, 2011, and US Provisional Application No. 61 / 449,882. All of the above are included as references.

The present application is based on the discovery and characterization of V-region immunoglobulin-containing T cell activation (VISTA Immunoglobulin-containing Suppressor of T cell Activation (VISTA) or PD-L3) It relates to hematopoieticaily-restricted and structurally distinct Ig-superfamily inhibitory ligands. The extracellular domain has PD-L1 homology to the B7 family ligand, and like PD-L1, VISTA has many effects on the immune system. However, unlike PD-L1, the expression of VISTA is only seen in the hematopoietic compartment. Expression is also interesting in subpopulations of CD4 + T cells and Foxp3 + regulatory T cells (Foxp3 + regulatory T cells; Treg) but predominantly predominantly in myeloid antigen presenting cells (APCs). Expression of soluble VISTA-Igs fusion proteins or VISTA in APCs strongly inhibits T cell proliferation, cytokine production in vitro and induces Foxp3 expression in T cells. In contrast, the newly developed anti-VISTA monoclonal antibody inhibits VISTA-induced immunosuppression of T cells by VISTA + APCs in vitro. In addition, in vivo anti-VISTA promotes the development of experimental allergic encephalomyelitis (EAE) with T-cell mediated autoimmune disease, and provides a protective, tumor-specific immune response with subsequent tumor remission . Early studies of VISTA - / - mice show early signs of spontaneous inflammatory disease and their pathological fate will be determined. Unlike all other PD-ligand-related molecules (e.g., B7-H3, H4, H6), the hematopoietic restriction of VISTA and its many inhibitory activities and inherent structural properties make VISTA a novel, , A negative regulator of immunity, whose expression is usually myeloid-restricted.

Induction of the immune response requires expansion, differentiation, contraction of T cells and generation of T cell memory (T cell memory). T cells must meet antigen presenting cells (APCs) and communicate through the major histocompatibility complex (MHC) interactions of T cell receptors / APCs. When a TCR / MHC mutual bond is generated, the binding of other receptor-ligands between T cells and APCs, such as co-stimulation via CD154 / CD40 and CD28 / B7.1-B7.2, is required. The synergy between these contacts results in a situation that can induce productive immune responses and autoimmunity that can remove pathogens and tumors in vivo.

Other levels of regulation have been identified, regulatory T cells (Treg). A specific subset of the T cells can be generated in the thymus, move to the periphery, and induce T cell response regulation constantly in vitro and in vivo (Sakaguchi (2000) Cell 101 (5): Rev. Immunol 18: 423-49; Bluestone and Abbas (2003) Nat. Rev. Immunol.3 (3): 253-7). Tregs express the CD4 + CD25 + phenotype and are also associated with cytotoxic T lymphocyte-associated antigen-4, OX-40, 4-1BB and glucocorticoid-inducible TNF receptor-associated (2002) Immunity 16 (2): 31 1-23; Shimizu, et al. (2002) Nat. Immun. 3 (2): 135-42 ). Removal of Treg cells by 5 day neonatal thymectomy or antibody depletion with an anti-CD25 antibody resulted in the induction of autoimmune pathogens and the induction of foreign and autologous tumors including anti- (1985) J. Immunol. 161 (1): 72-87; Sakaguchi, et al. (1995) J. Immunol 155 (3): 1151-64; Jones, et al. (2002) Cancer Immun. 2: 1). In addition, Tregs have been shown to be associated with poor tolerance to transplantation tolerance (Jarvinen, et al. (2003) Transplantation 76: 1375-9), since depletion of Tregs using anti-CD25 monoclonal antibodies results in the elimination of graft resistance and rapid rejection (2001) J. Immunol 166 (6): 3789-37%; Wood and Sakaguchi (2003) Nat. Rev. Immunol. 3: 199-210 ). Among the receptors expressed by Treg, ligation of GITR in the Treg surface using an agonistic monoclonal antibody in vitro or in vivo causes the termination of rapid Treg activity (McHugh, et (2002)), GITR is also an important component, since it also causes autoimmune pathology (Shimizu, et al. (2002)) and removal of transplant tolerance Respectively.

Costimulatory and co-inhibitory ligands and receptors not only provide a secondary signal for T cell activation, but also limit immune responses to self, while immunologic responses to infection And balances the network of positive and negative signals to maximize. The best characterized comet stimulating ligands are B7.1 and B7.2, which are expressed by professional APCs (professional APCs) and their receptors are CD28 and CTLA-4 (Greenwald, RJ, Freeman, GJ, and Sharpe, AH (2005) Annu Rev Immunol 23, 515-548; Sharpe, AH, and Freeman, GJ (2002) Nat Rev Immunol 2, 1 16- 126). CD28 is important for T cell activation and optimized by naive and activated T cells. On the other hand, CTLA-4 is induced after T cell activation and inhibits T cell activation by binding to B7.1 / B7.2, thus impairing CD28-mediated co-stimulation. CTLA-4 also converts negative signaling through its cytoplasmic motif (Teft, WA, Kirchhof, MG, and Madrenas, J. (2006). Annu Rev Immunol 24, 65-97 ; Teft, WA, Kirchhof, MG, and Madrenas, J. (2006) Annu Rev Immunol 24, 65-97). B7.1 / B7.2 KO mice exhibited an impaired adaptive immune response (Borriello, F., Sethna, MP, Boyd, SD, Schweitzer, AN, Tivol, EA, Jacoby, Freeman, GJ, Borriello, F., Hodes, RJ, Reiser, H., Hathcock, KS, Laszlo (1997) Immunity 6, 303-313; TB, Simpson, E. M, Freeman, GJ, and Sharpe, (1993), Science 262, 907-909), CTLA-4 KO mice were able to control inflammation as appropriate (1999) Immunity 7, 885-895; Tivol, EA, Borriello, F., Schweitzer, AN, Lynch, WP, Bluestone, JA, and Sharpe, AH (1995) Immunity 3, 541-547; Waterhouse, P., Penninger, JM, Timms, E., Wakeham, A., Shahinian, A., Lee, KP, Thompson, Griesser, H., and Mak, TW (1995), Science 270, 985-988).

B7 family ligands bind to the covalent B7-H2 (ICOS ligand) and B7-H3 as well as the co-inhibitors B7-H1 (PD-L1), B7-DC ), And B7-H6 (Brandt, CS, Baratin, M., Yi, E., Kennedy, J., Gao, Z., Fox, B., Haldeman, B., Ostrander, (2005) Annu Rev Immunol 23, 515-548), which is known to be involved in the pathogenesis of Parkinson's disease, .

Inducible co-stimulation (ICOS) molecules are expressed in activated T cells and bind to B7-H2 (Yoshinaga, S. K Whoriskey, JS, Khare, SD, Sarmiento, U., Guo, J., Horan, T. , Shih, G., Zhang, M., Coccia, MA, Kohno, T et al (1999) Nature 402, 827-832). ICOS is not only important for T cell activation, differentiation and function, but also for T-helper-cell-induced B cell activation, Ig class switching and germinal (2001) Nature 409, 97-101; Tafuri, R., et al., " Duncan, G., et al. (2001), " Shahinian, A., Bladt, F., Yoshinaga, SK, Jordana, M., Wakeham, A., Boucher, LM, Shah, G., Zhang, M., Coccia, MA, Kohno, K., " Nature 409, 105-109; Yoshinaga, SK, Whoriskey, JS, Khare, SD, Sarmiento, U., Guo, J., Horan, T., T., et al. (1999) Nature 402, 827-832). On the other hand, PD-1 (Programmed Death 1) is a negative control of T cell responses. PD-1 KO mice develop autoimmune dilated cardiomyopathy which is dependent on lupus-like autoimmune disease or genetic background (Nishimura, H., Nose, M., Hiai, H., Minato, N., and Honjo, T. (1999) Immunity 1, 141- 151. Nishimura, H., Okazaki, T., Tanaka, Y., Nakatani, K., Hara, M., Matsumori, A., , S., Mizoguchi, A., Hiai, H., Minato, N., and Honjo, T. (2001) Science 291, 319-322). Autoimmunity is usually caused by signal deletion by two ligands, PD-L1 and PD-L2. Recently, CD80 has been identified as a secondary receptor for PD-L1 that converts inhibitory signals to T cells (Butte, MJ, Keir, ME, Phamduy, TB, Sharpe, AH, and Freeman, GJ , 1 1 1 -122). The receptors for B7-H3 and B7-H4 are not yet known.

The most well known co-stimulatory ligands are B7.1 and B7.2, which belong to the Ig superfamily composed of a number of important immunomodulators, such as B7-based ligands and receptors. Ig counterparts are expressed in specialized antigen-presenting cells (APCs), and their receptors are CD28 and CTLA-4. CD28 is important for T cell activity that is expressed and optimized by naive and active T cells. On the other hand, CTLA-4 is induced following T cell activation and inhibits T cell activation by impairing CD28-mediated co-stimulation by inhibiting B7.1 and B7.2. B7.1 and B7.2 knockout (KO) mice show impaired adaptive immune responses, while CTLA-4 KO mice are unable to adequately control inflammation and develop systemic autoimmune diseases. Over time, the B7-based ligands exhibited co-stimulation such as B7-H2 (ICOS ligand) and B7-H3 and B7-HI (PD-L1), B7- , And co-inhibiting ligands such as B7-H6. Thus, additional CD28 family receptors have been identified. ICOS is expressed in activated T cells and binds to B7-H2. ICOS is a benign co-regulator and is important for T cell activation, differentiation and function. On the other hand, PD-1 (programmed death 1) negatively regulates T cell responses. PD-1 KO mice develop lupus-like autoimmune diseases or dilated cardiomyopathy. Contrary to VISTA (an immunosuppressive molecule that is the focus of the present invention), PD-L1 and PD-L2, two inhibitory B7 ligands, have distinct distinctive expression patterns. PD-L2 is expressed in DCs and macrophages, whereas PD-L1 is generally expressed in both hematopoietic cells and nonhematopoietic cells. Consistent with the immunosuppressive role of PD-1 receptors, studies using PD-L1 - / - and PD-L2 - / - mice showed that both ligands play a redundant role in inhibiting T cell proliferation and cytokine production . PD-L1 deficiency promotes disease progression in a non-obese diabetic model of autoimmune diabetes and a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis (EAE)). PD-L1 - / - T cells produce elevated levels of proinflammatory cytokines in these two disease models. In addition, studies in NOD mice have shown that tissue expression of PD-L1 (i.e., within the pancreas) is the only ability to localize inflammation. PD-L1 is also highly expressed in placental syncytiotrophoblasts, which is critical for controlling the maternal immune response to allogeneic fetuses.

Considering the enormous impact on the family of molecules that regulate immunity, considerable efforts with cancer mouse models have shown that protective anti-tumor immunity can be induced by targeting the family of molecules Can be. Studies involving anti-CTLA-4 have demonstrated enhanced therapeutic benefit in clinical studies of mouse models and melanomas. The mice vaccinated with B 16-GM-CSF (Gvax) increased the rejection of B 16 melanoma when used in combination with the blocking antibody of CTLA-4.

In addition, antibodies to PD-1 as well as PD-1 have enhanced enhanced anti-cancer immunity and host survival in a wide range of tumor models. Finally, although CTLA-4 and PD-1 belong to the same class of co-inhibitory molecules, evidence suggests that they use distinctly different T cell activity inhibition mechanisms and that anti-CTLA-4 and anti- There is a synergy effect in the ability to enhance host survival when PD-1 / Ll is coadministered.

Based on the above, it will be useful in the important role of these members in regulating clearance of another novel B7 type family member and ligand, and its modulators, in particular T cell immunity.

The present invention relates to blocking specific binding or binding of a specific regulatory T cell protein to its counterceptor. This protein, termed PD-L3 or VISTA, is a novel, structurally distinct, Ig-suppressive ligand and its extracellular domain is the B7 family ligand PD-L1 (B7 family ligand PD-L1). Wherein the molecule is referred to interchangeably as PD-L3 or VISTA or as V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) of T cell activity. VISTA is expressed primarily in hematopoietic components and is highly expressed in myeloid APCs (myeloid APCs) and T cells. Therapeutic modulation of the VISTA inhibitory pathway represents a novel approach to controlling T cell-mediated immunity for the treatment of a wide variety of cancers.

In particular, the invention relates to the use of antibodies specific for VISTA or PD-L3 to treat specific cancers, including bladder cancer, ovarian cancer, and melanoma.

In particular, the present invention also relates to the use of pf PD-L3 or a VISTA protein, especially a multimeric VISTA, and a viral vector expressing the same to treat a disease (for example, adenoviral) Wherein the immunosuppression is therapeutically desirable, for example allergic, autoimmune and inflammatory diseases.

As described below, expression of VISTA appears to be restricted to hematopoietic constituents, which are highly expressed in mature myeloid cells (CD1 l bbrighl) and expressed at lower levels in CD4 + T cells, Treg and CD8 + T cells . For example, soluble VISTA proteins such as soluble VISTA-Ig fusion proteins, or VISTA expression of APCs inhibit CD4 + and CD8 + T cell proliferation and cytokine production in vitro. In addition, we have observed, for example, that anti-VISTA antibodies, such as anti-VSTA mAb (13F3), block the inhibition of VISTA-induced T cell responses by VISTA + APCs in vitro. It has also been found that anti-VSTA mAbs exacerbate EAE in vivo and increase the frequency of encephalitogenic Th17s (encephalitogenic Th17s). Moreover, as described in the detailed description below, anti-VSTA mAbs have been found to induce tumor remission in many (4) mouse tumor models. In this model, expression of VISTA in myeloid derived suppressor cells (MDSC) is very high, which means that VISTA + MDSC inhibits tumor-specific immunity. As shown here, VISTA is an important regulator of immunosuppressive activity of T cells in vitro and in vivo in mice and humans (in vitro) and is an important regulator of autoimmune development and immune response to cancer. In particular, the data show:

(1) VISTA is a new member of the Ig superfamily and contains the Ig-V domain that is distant from PD-L1. Wherein VISTA inhibits mouse and human CD4 + and CD8 + T cell proliferation and cytokine production when produced as an Ig fusion protein or when overexpressed in artificial APCs (artificial APCs).

(2) VISTA expression of myeloid APCs is inhibitory to T cells in vitro.

VISTA expression of MDSC is very high in the tumor microenvironment. Previously, the phenotypic and functional analysis of many cell surface molecules suggests that they are associated with MDSC-mediated inhibition of T cells: CD115, CD124, CD80, PD-L1 and PD-L2 were expressed by MDSC, Expression levels or percentage levels are not found to be heterogeneous between MDSCs and cells from tumor-free mice lacking immunosuppressive activity. Therefore, it is expected that VISTA will be a major B7 negative modulator in MDSC.

(4) Antibody-mediated VISTA blockade leads to protective immunity to autologous tumors.

Based on the above, VISTA appears to be a predominant negative immunomodulatory molecule in MDSC, involving the development of protective anti-tumor immunity. Therefore, blocking the activity of this molecule using anti-VSTA antibodies will allow the development of protective anti-tumor immunity in humans and other mammals.

Therefore, the present invention relates to the use of VISTA as an immunomodulatory agent in the treatment or prevention of VISTA and its use in the treatment of different cancers such as bladder cancer, ovarian cancer and lymphoma, and autoimmune diseases, allergies, infections and inflammatory diseases A soluble VISTA protein, including multiple copies of VISTA extracellular domain or a fragment thereof, and a VISTA binding agent, such as small molecules and antibodies or fragments thereof, for example, for multiple sclerosis and arthritis, , Such as fusion proteins, and the use of multimeric VISTA proteins.

As described in the detailed description below, this protein is a novel inhibitory ligand that has extracellular Ig-V domains homologous to two known B7 based ligands.

PD-L1 and PD-L2 (Programmed Death Ligands 1 and 2) exhibit unique sequence characteristics and exhibit unique expression patterns in APCs and subpopulations of T cells in vitro and in vivo Or VISTA). This protein has a functional effect on CD4 + and CD8 + T cell proliferation and differentiation (inhibits CD4 + and CD8 + T cell proliferation as well as cytokine production). Based on its expression pattern and its inhibitory effect on T cells, PD-L3 or VISTA during the cognate interaction between T cells and bone marrow-derived APCs is regulatory ligand that negatively regulates T cell responses ).

PD-L3 or VISTA was shown to be a member of the B7 family ligand, unlike other B7 family ligands, this molecule contained only the Ig-V domain without the Ig-C domain and phylogenetically, the B7 family receptor PD- (Programmed Death-1). In contrast, PD-L3 or VISTA, and their specific agonists or antagonists, regulate T cell activation and differentiation, and more broadly regulate a regulatory network that regulates the immune response. Can be used to. In particular, antibodies specific for PD-L3 or VISTA proteins and PD-L3 or VISTA agonists or antagonists, preferably PD-L3 or VISTA, are used in the treatment of autoimmune, inflammatory reactions and diseases, allergies, cancers, infectious diseases and transplantation, Lt; / RTI >

Thus, the present invention is based, in part, on the use of isolated soluble PD-L3 or VISTA proteins, or soluble VISTA-Ig fusion proteins, or multimeric VISTA proteins, for use in compositions, e. Fusion protein, wherein preferably at least 70-90% identical to the amino acid sequence of human or mouse PD-L3 or VISTA polypeptide or ortholog described in SEQ ID NO: 2, 4 or 5 , Or a fragment thereof encoded by a gene that specifically hybridizes to SEQ ID NO: 1 or 3 that regulates VISTA in vivo and a pharmaceutically acceptable carrier. In some embodiments, a soluble or multimeric VISTA protein can be directly or indirectly linked to a heterologous (non-VISTA) or viral vector or, for example, a transfected, immune cell, such as a T cell ≪ / RTI >

The present invention also relates to an expression vector comprising an isolated nucleic acid encoding at least 70-90% of the VISTA protein or fragment or its heterologous foci, which is at least 70-90% identical to the human or mouse VISTA amino acid sequence set forth in SEQ ID NO: 2, 4 or 5 And a host cell comprising the vector, wherein the vector is optionally fused with a sequence encoding an Ig polypeptide, such as an Fc region or another protein, such as a receptor molecule.

The present invention also relates to a specific binding agent, preferably a PD-L3 or VISTA protein comprising an amino acid sequence or variant, a fragment or an isoform thereof as disclosed in SEQ ID NO: 2, 4 or 5 Antibody fragment or fragment thereof. In a preferred embodiment, the binding agent modulates (acts or antagonizes) VISTA activity in vitro or in vivo. In most preferred embodiments, the binding agent is an agonistic or antagonistic antibody.

The present invention further provides a method for modulating immune cell response by contacting said immune cell with a VISTA protein or a binding agent that specifically binds to said immune cell in vitro or in vivo in the presence of a primary signal to modulate immune cell response ≪ / RTI > (rVISTA EH binds its regulator to the immune cells and regulates the immune response.) The PD-L3 or VISTA protein binds to the myeloid lineage, including dendritic cells (DCs) and macrophages PD-L3 and VISTA expression is up-regulated in myeloid APCs, but not in CD4 + T cells, after immunostimulation. The expression of PD-L3 and VISTA is upregulated in myeloid antigen presenting cells, Cells. Thus, PD-L3 or VISTA nucleic acids and polypeptides of the invention, and agonists or antagonists thereof, are useful, for example, in modulating the immune response.

Another aspect of the invention is the use of primers for the detection of PD-L3 or VISTA-encoding nucleic acids, as well as isolated nucleic acid fragment molecules, preferably encoded soluble fusion proteins and polymorphic VISTA proteins, encoding the VISTA polypeptide. Or hybridization probes. ≪ / RTI > In some embodiments, a PD-L3 or VISTA nucleic acid molecule of the invention encodes a PD-L3 or VISTA of SEQ ID NO: 1 or 3, or a nucleotide sequence encoding at least about 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

In yet another embodiment, the PD-L3 or VISTA nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence having a specific percent identity with the amino acid sequence of SEQ ID NO: 2, 4 or 5 . In certain preferred embodiments, the PD-L3 or VISTA nucleic acid molecule is at least about 71%, 75%, or at least about 70% identical to the entire amino acid sequence of SEQ ID NO: 2, 4 or 5 and / or its extracellular domain. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of the polypeptide.

In another preferred embodiment, the isolated nucleic acid molecule encodes the amino acid sequence of a conserved or functional domain of VISTA in or of a human or mouse. In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4 or 5. In another preferred embodiment, the length of the nucleic acid molecule is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more nucleotides. In another preferred embodiment, the length of the nucleic acid molecule is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more nucleotides and the nucleic acid encodes a polypeptide having PD-L3 or VISTA activity or modulating PD-L3 or VISTA function (described herein).

In yet another embodiment of the present invention, a nucleic acid molecule, preferably a PD-L3 or VISTA nucleic acid molecule, is characterized, which specifically relates PD-L3 or VISTA It detects nucleic acid molecules. For example, in some embodiments, the length of the nucleic acid molecule is at least about 880, 900, 950, 1000, 1050, 1100, 1150 or more nucleotides, : 2, 4 or 5, or a nucleic acid molecule encoding the complementary sequence thereof. In yet another embodiment, the length of the nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300 or more nucleotides, L3 or VISTA such as at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950 or more nucleotides. Said nucleic acid comprises at least 15 (i.e., consecutive 15) or more of the nucleic acid sequences disclosed in SEQ ID NO: 2, 4 or 5, or SEQ ID NOS: 1 and 3 encoding the complementary sequence PD-L3 or VISTA polypeptide And hybridizes to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a complementary sequence thereof, under certain conditions.

In another preferred embodiment, the nucleic acid molecule encodes a naturally occurring allele variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4 or 5, wherein the nucleic acid molecule encodes a polypeptide having the sequence SEQ ID NO: 1, 3, or a complementary sequence thereof.

Yet another embodiment of the present invention is directed to an antisense for a PD-L3 or VISTA nucleic acid molecule, for example a coding strand of a PD-L3 or VISTA nucleic acid molecule as shown in SEQ ID NO: 1 or 3, , ≪ / RTI > isolated nucleic acid molecule.

Another aspect of the invention provides vectors comprising PD-L3 or VISTA nucleic acid molecules. In some embodiments, the vector is a recombinant expression vector.

In another embodiment, the invention provides a host cell comprising a vector of the invention. In another embodiment, the invention provides a host cell comprising a nucleic acid of the invention. The present invention also provides a method for producing a polypeptide of the present invention comprising culturing a mammalian host cell of the present invention comprising a recombinant expression vector such as a host cell, for example, a non-human mammalian cell, preferably a PD-L3 or VISTA polypeptide by culturing the above polypeptide.

Another aspect of the invention is characterized by isolated or recombinant PD-L3 or VISTA polypeptides (e. G., Proteins, polypeptides, peptides or fragments or portions thereof). In one embodiment, the isolated PD-L3 or VISTA polypeptide or PD-L3 or VISTA fusion protein comprises one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, A transmembrane domain, and a cytoplasmic domain.

In a preferred embodiment, the PD-L3 or VISTA polypeptide comprises one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. And at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity with the amino acid sequence of SEQ ID NO: , 98%, 99% or more. In another preferred embodiment, the PD-L3 or VISTA polypeptide comprises one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. And VISTA or PD-L3 activity (as described herein).

In yet another embodiment, the PD-L3 polypeptide comprises one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. And under the hybridization specific conditions, is encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes to a complementary sequence of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3.

In another embodiment, the invention features a fragment or portion of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4 or 5, wherein said fragment comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4 And 15 amino acids (i.e., consecutive amino acids) of the amino acid sequence. In yet another embodiment, the PD-L3 or VISTA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, 4 or 5. In yet another embodiment, the invention provides a polynucleotide comprising a nucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% identical to the nucleotide sequence of SEQ ID NO: L3 or VISTA polypeptide encoded by a nucleic acid molecule consisting of a nucleotide sequence that is at least 95%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence. Further, the present invention is characterized by a PD-L3 or VISTA polypeptide encoded by a nucleic acid molecule consisting of a nucleotide sequence hybridized with a complementary sequence of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3 under hybridization specific conditions.

The polypeptide of the present invention or a portion thereof, e.g., a biologically active portion thereof, is operatively linked to a non-PD-L3 (non-PD-L3) or VISTA polypeptide (e. G., A heterologous amino acid Sequence) to form a fusion polypeptide. Furthermore, the invention features an antibody, for example a monoclonal antibody or a polyclonal antibody, which specifically binds to a polypeptide of the invention, preferably a human PD-L3 or VISTA polypeptide .

The present invention also relates to the use of the compounds of the present invention for the production of the desired functional properties such as the inhibitory effect of the present protein on TCR activation, the inhibitory effect of CD4 + T cell proliferation on anti-CD3, the effect of cognate CD4 + T cells, L3 or VISTA in the expression of specific cytokines such as IL-2 and gamma interferon, as well as inhibition of antigen-specific proliferation of PD-L3 PD-L3 or VISTA antibodies having desirable functional properties from a population of monoclonal antibodies produced against said protein or PD-L3 or VISTA-Ig fusion protein based on the property of modulating the specific effect of VISTA or VISTA . In certain preferred embodiments, for therapeutic use, the anti-PD-L3 or VISTA antibody is administered in the presence of an in vitro soluble PD-L3 or VISTA-protein, for example in the presence of a PD-L3 or VISTA fusion protein, Will be screened to promote the inhibitory effect of PD-L3 or VISTA-Ig in the relevant PD-L3 or VISTA. In immunity, somewhat unexpectedly (see below), these antibodies are expected from their effects in vivo in vitro, i.e., because anti-or VISTA monoclonal antibodies work against the immunosuppressive properties, this is desirable .

In addition, the PD-L3 or VISTA polypeptide (or a biologically active portion thereof) or a modulator of the PD-L3 or VISTA molecule, i.e. an antibody such as selected using the method, , Which includes pharmaceutically acceptable carreiers.

In yet another embodiment, the PD-L3 or VISTA protein is used as an inhibitory signal to reduce or inhibit immune cell activation. In such an embodiment, the inhibitory signal is associated with an activating receptor (e. G., TCR, < RTI ID = 0.0 > CD3, BCR, or Fc polypeptide). Inhibition may be, for example, inhibition of secondary messenger formation; Inhibition of proliferation; Inhibition of the effector function of immune cells, such as reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, mediators (e. G., Cytokines (IL-2) and / or the mediator of the allergic response); Or the development of anergy.

In some embodiments, the primary signal is a ligand (e. G., CD3 or anti-CD3) that binds to the TCR and initiates a primary stimulation signal. The TCR ligand is commercially available and specific examples include anti-CD3 antibody OK.T3 and anti-CD3 monoclonal antibody G1-CD3 prepared from hybridoma cells obtained from the American Type Culture Collection, 4. In another embodiment, the primary signal is a protein kinase C activator, such as a phorbol ester (e.g., phorbol myristate acetate) Is delivered to T cells through other mechanisms including calcium ionophore (e. G., Ionomycin, which increases cytoplasmic calcium concentration), or the like. The use of the agent bypasses the TCR CD3 complex, but delivers stimulatory signals to T cells. Other agents that act as primary signals may include natural and synthetic ligands. Natural ligands may include MHC with or without peptides. Other ligands include peptides, polypeptides, growth factors, cytokines, chemokines, glycopeptides, soluble receptors, steroids, hormones, mitogens such as PHA or other superantigen, peptide- MHC 4 tetramer (Altman, et al. (1996) Science 274 (5284): 94-6) and soluble MHC 2 dimer (Dal Porto, et al (1993) Proc Natl. USA 90: 6671-5).

The activated immune cells according to the method of the present invention can then be expanded ex vivo and used in the treatment and prevention of various diseases: for example, human T cells are cloned in vitro and expanded to produce modulatory activity (Groux, et al. (1997) Nature 389 (6652): 737-42). Prior to expansion, the source of the T cells is obtained from a human, dog, cat, mouse, rat, or a genetic variant thereof, and the T cells are peripheral blood mononuclear cells , Bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissues from infected areas, spleen tissues, tumors or T cell lines. T cells can be obtained from the blood obtained from an individual using any technique well known to those skilled in the art, e.g., Ficoll ™ seperation.

In yet another aspect, the present invention provides a method for detecting a PD-L3 or VISTA nucleic acid molecule, protein or polypeptide by contacting the biological sample with a PD-L3 or VISTA nucleic acid molecule, a protein or polypeptide capable of detecting the polypeptide, To detect the presence of PD-L3 or VISTA nucleic acid molecules, proteins or polypeptides in a biological sample. Such PD-L3 or VISTA expression can be used to detect specific disease sites, such as inflammatory sites.

In another aspect, the invention provides a method for detecting the presence of PD-L3 or VISTA activity in a biological sample by contacting said biological sample with an agent capable of detecting an indicator of PD-L3 or VISTA activity To detect the presence of PD-L3 or VISTA activity in the biological sample.

In another aspect, the present invention provides a method for modulating PD-L3 or VISTA activity, comprising contacting a cell capable of expressing PD-L3 or VISTA with an agent capable of modulating PD-L3 or VISTA activity, PD-L3 < / RTI > or VISTA activity can be modulated, including contacting the cell with an anti-PD-L3 or VISTA antibody. In some embodiments, the agent inhibits PD-L3 or VISTA activity. In another embodiment, the agent stimulates PD-L3 or VISTA activity. Further, in another embodiment, the agent enhances or prevents binding between the PD-L3 or VISTA polypeptide and its natural binding partner. In one embodiment, the agent is an antibody that specifically binds a PD-L3 or VISTA polypeptide. In another embodiment, the agent is a peptide, peptidomimetic or other small molecule that binds to a PD-L3 or VISTA polypeptide.

In yet another embodiment, the agent modulates PD-L3 or VISTA gene transcription, PD-L3 or VISTA mRNA translation, or post-translational modification of PD-L3 or VISTA polypetide, -L3 or VISTA. ≪ / RTI > In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to a coding strand of PD-L3 or VISTA mRNA or PD-L3 or VISTA gene.

In certain embodiments, the methods of the invention comprise administering an individual having a disorder or condition characterized by an aberrant, insufficient or unwanted PD-L3 or VISTA polypeptide or nucleic acid expression or activity, For treatment, PD-L3 or VISTA modulators are used by administering to the subject. In certain preferred embodiments, the PD-L3 or VISTA modulator is a PD-L3 or VISTA polypeptide, preferably a soluble fusion protein or multimer VISTA protein or anti-VSTA antibody described below. In yet another embodiment, the PD-L3 or VISTA modulator is, for example, a PD-L3 or VISTA nucleic acid molecule in an adenoviral vector. In another embodiment, the invention provides treating the subject with an additional agent that modulates the immune response.

In another embodiment, the invention provides a vaccine comprising an antigen and an agent that modulates (enhances or inhibits) PD-L3 or VISTA activity. In a preferred embodiment, the vaccine inhibits mutual binding between PD-L3 or VISTA and its natural binding partner.

The invention also provides a diagnostic test for identifying the presence or absence of a genetic alteration characterized by one or more of the following: (i) an abnormal variation of a gene encoding a PD-L3 or VISTA polypeptide Or mutations; (ii) misregulation of the gene; And (iii) an abnormal post-translational modification of a PD-L3 or VISTA polypeptide, wherein the wild type of said gene encodes a polypeptide having PD-L3 or VISTA activity.

In another aspect, the invention provides an indicator composition comprising PD-L3 or VISTA polypeptide having PD-L3 or VISTA activity, contacting the test compound with the indicator composition, and PD-L3 or < RTI ID = A compound that binds to or modulates PD-L3 or VISTA polypeptide by determining the effect of the test compound on PD-L3 or VISTA activity in an indicator compound to identify a compound that modulates the activity of the VISTA polypeptide A method for identification is provided.

In some aspects, the invention features a method for modulating the interaction of PD-L3 or VISTA with its natural binding partner in immune cells, comprising contacting PD-L3 or VISTA with a formulation selected from the group consisting of PD3 or VISTA, or PD-L3 or VISTA in the form of an agent that modulates interactions with PD-L3 or VISTA and its natural binding partner, The mutual coupling of natural binding partners can be controlled. In a preferred embodiment, the agent that modulates the interaction of PD-L3 or VISTA with its natural binding partner is an antibody that specifically binds PD-L3 or VISTA. In one embodiment, the mutual coupling of PD-L3 or VISTA and its natural binding partner is up-regulated. In another embodiment, the mutual coupling of PD-L3 or VISTA and its natural binding partner is down-regulated. In certain embodiments, the method comprises contacting the immune cell or antigen presenting cell with an additional agent that modulates the immune response.

In certain embodiments, the step of contacting is performed in vitro. In certain embodiments, the step of contacting is performed in vivo. In certain embodiments, the immune cells are selected from the group consisting of: T cells, monocytes, macrophages, dendritic cells, and myeloid cells.

In another embodiment, the present invention relates to a method for inhibiting or increasing activation in immune cells, including increasing or inhibiting the expression or activity of PD-L3 or VISTA, and inhibiting the activity of immune cells Or may be increased.

In another aspect, the invention is directed to a vaccine comprising an agent and an agent that inhibits the interaction between the antigen and PD-L3 or VISTA and its natural binding partner.

In another aspect, the invention relates to a vaccine comprising an agent and an agent that promotes cross-linking between the antigen and PD-L3 or VISTA and its natural binding partner.

In another aspect, the present invention relates to a method for treating a subject having a disease that may benefit from upregulation of the immune response, comprising administering to a subject in need thereof a therapeutically effective amount of PD-L3 or VISTA, Comprising administering an agent that inhibits mutual binding between the binding partners, thereby treating a disease that may benefit from upregulation of the immune response. In a preferred embodiment, the agent comprises a blocking antibody or small molecule that binds PD-L3 or VISTA and inhibits cross-linking between PD-L3 or VISTA and its natural binding partner. In another embodiment, the method comprises administering to the subject a second agent that upregulates the immune response. In another aspect, the present invention relates to a method of treating a subject having a disease that may benefit from down-regulation of an immune response, wherein the interaction between PD-L3 or VISTA and its natural binding partner Comprising administering an agent that stimulates the binding, whereby a disease that may benefit from downregulation of the immune response can be treated.

For example, diseases that are treated with a PD-L3 or VISTA protein or a binding agent are selected from the group consisting of: a tumor, a pathogenic infection, an inflammatory immune response or disease, preferably a less specific inflammatory disease, Immunosuppressive disease. Specific examples include two types of advanced and early types of cancer, including multiple sclerosis, thyroiditis, rheumatoid arthritis, diabetes Type II and I, and metastatic cancers, including bladder cancer, ovarian Cancer, melanoma, lung cancer, and other cancers where VISTA inhibits an effective anti-tumor response. In some instances, each individual may be administered a cell or viral vector expressing the nucleic acid encoding the anti-VISTA antibody or VISTA fusion protein.

In certain embodiments, the agent comprises an antibody or small molecule that stimulates a mutual binding between PD-L3 or VISTA and its natural binding partner. In another embodiment, the method comprises administering to the subject a second agent that downregulates the immune response, such as a PD-L !, PD-L2 or CTLA-4 fusion protein or an antibody specific thereto.

For example, exemplary diseases that can be treated using the PD-L3 or VISTA proteins, binders or PD-L3 or VISTA agonists or antagonists according to the present invention are transplant, allergy, infectious disease, cancer, and inflammatory or autoimmune disorders such as inflammatory immune disorders. Specific examples of such diseases include type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosis, rheumatoid disease such as rheumatic diseases, allergic disorders, asthma, allergic rhinitis, skin disorders, Crohn's disease, and ulcerative colitis. gastrointestinal disorders, transplant rejection, post-streptococcal infection and poststreptococcal and autoimmune renal failure, septic shock, systemic inflammatory response syndrome (SIRS) Adult respiratory distress syndrome (ARDS) and animal poisoning (envenomation); Degenerative bone and joint diseases, including osteoarthritis, crystal arthritis and capsulitis, and other arthropathies, as well as autoinflammatory diseases. Further, the methods and compositions can be used to treat tendonitis, ligaments and traumatic joint injuries.

In another aspect, the present invention relates to a cell-based assay for screening compounds that modulate the activity of PD-L3 or VISTA, comprising contacting cells expressing a PD-L3 or VISTA target molecule And determining the ability of the test compound to modulate the activity of the PD-L3 or VISTA target molecule.

In yet another embodiment, the present invention relates to a cell-free assay for screening compounds that modulate binding of PD-L3 or VISTA to a target molecule, wherein PD-L3 or VISTA polypeptide or a Contacting the biologically active portion with a test compound and determining the ability of the test compound to bind to the PD-L3 or VISTA polypeptide or a biologically active portion thereof.

In yet another embodiment, the invention provides an anti-PD-L3 or VISTA antibody that modulates the effect of PD-L3 or VISTA on T cell activation and cytokine production at a compound, e. G., Primary and secondary antigen concentration Comprising contacting a test compound with a T cell expressing a PD-L3 or VISTA target molecule at a primary antigen concentration, contacting the test compound with a test compound that modulates T cell proliferation or cytokine production at the primary antigen concentration, Determining the ability of the compound to contact the T-cell expressing a PD-L3 or VISTA target molecule at a secondary antigen concentration with a test compound, contacting the T-cell expressing the PD-L3 or VISTA target molecule with the test compound at the secondary antigen concentration, Determining the ability of the test compound to thereby identify compounds that modulate T cell activation or cytokine production at primary and secondary antigen concentrations.

In another specific embodiment, anti-PD < RTI ID = 0.0 > (PD) < / RTI > is selected to inhibit or promote PD-L3 or VISTA effects in differentiation, proliferation, and / or cytokine production of CD4 + and CD8 + T cells in vitro or in vivo -L3 or VISTA antibodies and a population of PD-L3 or VISTA proteins can be screened.

In a preferred embodiment, each of the antibodies having a PD-L3 or VISTA protein, a nucleic acid and a ligand specific for PD-L3 or VISTA, preferably having a PD-L3 or VISTA function, is selected from the group consisting of cancer, autoimmune disease, Inflammatory disorders or infections and more specifically severe combined immunodeficiency, multiple sclerosis, systemic lupus erythematosus, type I diabetes mellitus, lymphoproliferative Lymphoproliferative syndrome, inflammatory bowel disease, allergies, asthma, graft-versus-host disease, and transplant rejection; Immune responses to infectious pathogens such as bacteria and viruses; And immune system disorders such as immune system cancers such as lymphomas and leukemia.

Figure 1. Sequence analysis. a. Amino route sequence of mouse PD-L3 or VISTA Full length. b. The amino acid sequence of the extracellular Ig domain between selected B7-type ligands including mouse PD-L3 or VISTA and B7-H1 (PD-L1), B7-DC (PD-L2), B7-H3, and B7- Alignment. c. Arrays of B7 family receptors including PD-L3 or VISTA Ig domains, PD-1, CTLA-4, CD28, BTLA, and ICOS. Ig-v domain, "...."; Ig-c domain, "" The array was performed using the MUSCLE algorithm (Multiple Sequence Comparison by Log-Expectation). d. Sequence identity (%) of the Ig-V domain between PD-L3 or VISTA and other B7 based ligands and receptors was calculated using the ClustalW2 program. e. Sequence homology between PD-L3 or VISTA in humans and mice. The same residue was shaded in black. The highly conserved and half-preserved residues were each shaded with dark color and light gray.
Figure 2. Systematic analysis of members of immunoglobulin (Ig) superfamily and other PD-L3 or VISTA mice. Mouse PD-L3 or VISTA, CD28, CTLA-4, ICOS, BTLA, PD-1. Including B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2, B7-H3, B7-H4, B7-1, B7-2, BTNL2, BTN3A3, BTN2A2, The sequence length of the Ig counterparts was analyzed using the PhyML algorithm (Phylogenetic Maximum Likelihood). Branch distances are shown at tree branch joints.
Figure 3. Tissue expression and hematopoietic cell expression patterns of PD-L3 or VISTA. a. RT-PCR of PD-L3 or VISTA battlefield from mouse tissue. Lane: (1) Muscle (2) Heart (3) Eye (4) Thymus (5) Spleen (6) Small intestine (7) Kidney (8) Liver (9) Brain (10) Mammalian gland (11) Lung (12) Ovary (13) Bone marrow. b. RT-PCR of PD-L3 or VISTA battlefield from purified hematopoietic cell types. Lanes: (1) peritoneal macrophages (2) spleen CD11b + monocytes (splenic CD1lb + monocytes) (3) spleen CD11c + DCs (splenic CD1lc + DCs) Splenic CD8 + T cells (splenic CD8 + T cells) (6) Splenic B cells. ce. Cellular flow analysis of PD-L3 or VISTA expression in the CD11c + DC subpopulation (e) from CD4 + and CD8 + T cells (c), CD11b + monocytes (d), spleen and peritoneal cavity of the spleen from the thymus and spleen (flow cytometry analysis). f. The splenic B cells, NK cells and granulocytes were also analyzed. g. Different expression of PD-L3 or VISTA in hematopoietic cells from mesenteric LN, peripheral lymph nodes, spleen, blood and abdominal cavity. Representative data from at least 3 independent experiments are shown
Figure 4. Gene array data of the PD-L3 or VISTA from the NCBI gene expression omnibus (GEO) database as well as the gene array database of the Genomics Institute of Novartis Research Foundation (GNF).
Figure 5. Specificity of PD-L3 or VISTA hamster monoclonal antibody. Mouse EL4 cell lines overexpressing PD-L1 or PD-L3 or VISTA are stained with supernatants from hybridoma cultures and analyzed by flow cytometry. Two representative positive clones are shown.
Figure 6. Comparison of PD-L3 or VISTA expression in cultured splenocytes in vitro with other B7 ligands. Comparison of the expression of PD-L3 or VISTA and other B7 ligands (i.e. PD-L1, PD-L2, B7-H3, and B7-H4) in hematopoietic cell types including CD4 + T cells, CD11bhi monocytes and CD11c + Respectively. Cells were either freshly isolated or cultured in vitro or in an inactive state for 24 hours in vitro. CD4 + T cells were activated with plate-bound CD3 (5 μg / ml) and CD11bhi monocytes and CD11c + DCs were activated with IFN alpha (20 ng / ml) and LPS (200 ng / ml).
Representative results are shown from three independent experiments.
Figure 7. In vivo expression pattern of PD-L3 or VISTA and other B7-based ligands during immunization. DO11.10 TCR transgenic mice were immunized with chicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA). Draining and non-draining lymph node cells were obtained 24 hours after immunization and analyzed by cell flow analysis for expression of PD-L3 or VISTA, PD-L1 or PD-L2. Representative results from at least 4 independent experiments are shown. a. In a draining lymph node, CD11b + cell populations expressing PD-L3 or VISTA at a high level were induced 24 hours after CFA / OVA immunization, but not CFA alone. These cells have a mixed phenotype of F4 / 80 + macrophages and CD11c + dendritic cells. b. Expression of PD-L3 or VISTA PD-L1 and PD-L2 of CDllbhi monocytes, CDllc + DCs and CD4 + T cells was analyzed after 24 hours of immunization.
Figure 8. Deletion of PD-L3 or VISTA in activated CD4 + T cells in response to immunization. DO11.10 mice were immunized laterally with chicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA). Drain and non-draining lymph node cells were obtained 48 hours after immunization and analyzed by cell flow analysis for expression of PD-L3 or VISTA. Representative results from at least two independent experiments are shown.
Figure 9. Fixed PD-L3 or VISTA-Ig fusion protein inhibits CD4 + and CD8 + T cell proliferation. a. CFSE-labeled CD4 + and CD8 + T cells are stimulated with or without PD-L3 or VISTA-Ig (co-absorbed PD-L3 OR VISTA-Ig) co-absorbed by plate-bound CD3. Percentages of CFSE-low cells are quantified and shown at b. c. CD4 + T cells from PD-1 KO mice are also inhibited by PD-L3 or VISTA-Ig. d. PD-L3 or VISTA-Ig-mediated inhibition is persistent and may work later. CD4 + T cells are activated for 72 hours (hr) (i), or for 24 hours (ii, iii and iv) in the presence of PD-L3 or VISTA-Ig or control-Ig. 24 hr-preactivated cells were obtained and re-stimulated under specific conditions for another 48 hours. Cell proliferation was analyzed at the end of the 72 hour incubation, (ii) pre-activation with PD-L3 or VISTA-Ig and re-stimulation with anti-CD3; (iii) Reactivation with CD3 and re-stimulation with PD-L3 or VISTA-Ig. (iv) pre-activated with PD-L3 or VISTA-Ig and re-stimulated with PD-L3 or VISTA-Ig. Duplicated wells were analyzed under all conditions. Representative results from at least 4 experiments are shown.
10. Similar inhibitory effects of PD-Ll-Ig and PD-L3 or VISTA-Ig fusion proteins in CD4 + T cell proliferation. Macro purified CD4 + T cells were CFSE labeled and stimulated with plate-bound CD3 with an appropriate amount of PD-L1-Ig or PD-L3 or VISTA-Ig fusion protein. CFSE dilution was analyzed at 72 hours and the percentage of CFSElow (CFSElow) cells was quantified. The double treated wells were analyzed under all conditions. Representative results from two experiments are shown.
Figure 11. Naive and memory inhibitory effects of PD-L3 or VISTA-Ig on proliferation of CD4 + T cells. (CD25-CD441owCD62Lhi) and memory (CD25-CD44hiCD62Llow) CD4 + T cell subpopulations were grouped and CFSE labeled and labeled with PD-L3 or VISTA-Ig or control- - conjugated anti-CD3 (2.5 g / ml). Cell proliferation was analyzed at 72 hours by examining the CFSE division profile. Percentages of proliferated cells, as determined by the percentage of CFSE low (CFSElow) cells, were calculated and shown in B. The double treated wells were analyzed under all conditions. Representative results from two experiments are shown.
Figure 12. PD-L3 or VISTA-Ig fusion protein inhibited early TCR activation and cell proliferation, but did not directly induce apoptosis. Macrofiltered CD4 + T cells were CFSE labeled and stimulated with plate-bound CD3 with 1-2 ratios of PD-L1-Ig or PD-L3 or VISTA-Ig fusion proteins (2.5 g ml and 5 g / ml, . CFSE dilution was analyzed by flow cytometry for expression of CD69, CD62L, and CD44 at 24 and 48 hours. Cells were also stained with the early apoptosis marker annexin-V and the cell death marker 7-Aminoactinomycin D (7-AAD). Representative results from two experiments are shown.
Figure 13. PD-L3 or VISTA-Ig inhibits cytokine production by CD4 + and CD8 + T cells. AB. Macrofiltered CD4 + T cells were CFSE labeled and stimulated with PD-L3 or VISTA-Ig or control-Ig, and plate-bound anti-CD3 at the indicated rates. Culture supernatants were collected after 24 and 48 hours. Levels of IL-2 and IFN? Were analyzed by ELISA. CD. CD4 + T cells were divided into naive (CD25-CD441owCD62Lhi) and memory (CD25-CD44hiCD62Llow) cell populations. Cells were stimulated with plate-bound CD3 and PD-L3 or VISTA-Ig or control-Ig at a ratio of 1: 2. Culture supernatants were harvested 48 hours later and analyzed by ELISA for levels of IL-2 and IFN □. E. Large purified CD8 + T cells were stimulated with PD-L3 or VISTA-Ig or control-Ig, and plate-bound CD3 in the indicated ratios. IFN □ in the culture supernatant was analyzed by ELISA. At all conditions, the supernatants of six duplicated wells were collected for ELISA analysis. Representative results obtained from at least 3 experiments are shown.
14. PD-L3 or VISTA-Ig-mediated inhibition can overcome the mid-level of co-stimulation provided by CD28 and can be partially rescued by exogenous IL-2, Lt; / RTI > ab. CD4 + T cells were activated by plate-bound CD3 with either PD-L3 or VISTA-Ig or control-Ig at 1-1 and 1-2 ratios. For the cytokine release, soluble mIL-2, mEL7, mIL15 and mIL-23 (all at 40 ng / ml) were added to the cell culture medium (a). To investigate the effects of co-stimulation, □ CD28 (1 μg / ml) was fixed at the ratios presented with CD3 and Ig proteins. (b). Cell proliferation was analyzed at 72 hours by examining the CESE cell division profile. CD. To investigate the inhibitory activity of PD-L3 or VISTA in the presence of low levels of co-stimulation, the titrated amount of CD28 was measured using anti-CD3 (2.5 μg / ml) in order to stimulate the proliferation of CD4 + And PD-L3 or VISTA-Ig fusion protein or control-Ig fusion protein (10 mu g / ml). Cell proliferation was analyzed at 72 hours. Percentages of proliferated CESElow cells were quantified and presented in d. The double treated wells were analyzed for all conditions. The results of representative CFSE profiles obtained from at least 3 experiments are shown.
Figure 15. PD-L3 or VISTA expressed in antigen presenting cells inhibits CD4 + T cell proliferation. ac. A CHO cell line stably expressing MHCII molecule I-Ad and co-stimulatory molecule B7-2 was used as a parent cell line. Retroviruses expressing either PD-L3 or VISTA-RFP or RFP control molecules were introduced into the cells. The introduced cells were sorted to achieve the same level of expression. In order to examine their antigen presenting cell capacity, CHO-PD-L3 or VISTA or CHO-RFP cells were treated with mitomycin C and expressed in OVA-specific transduced CD4 + T cells in the presence of an appropriate amount of OVA peptide 0.0 > D011. ≪ / RTI > The proliferation of DO11 was analyzed at 72 hours by CFSE cell division profile (ab) or by tritium absorption (c). d. During the 10 day incubation period, RFP or B7B-H5-RFP retrovirus was introduced into bone marrow-derived dendritic cells. The introduced CD11c + RFP + DCs and unincorporated CD11c + RFP-DCs were sorted and used in the presence of an appropriate amount of OVA peptide to stimulate OVA-specific transduced CD4 + T cells. Cell proliferation was analyzed at day 3 by examining CFSE cell division. For all experiments, duplicate wells were analyzed under all conditions and show representative results from at least 3 experiments.
Figure 16. Surface expression levels of PD-L3 or VISTA in bone marrow-derived DCs introduced as retroviruses. Bone marrow-derived DCs (BMDC) are cultured in the presence of GM-CSF (20 ng mml) and RFP or PD-L3 or VISTA-RFP retroviruses are introduced as described in the experimental method. On the tenth day, surface expression levels of PD-L3 or VISTA in cultured BMDCs are analyzed and compared to freshly-isolated peritoneal macrophages.
Figure 17 shows that the anti-PD-L3 mAb shows efficacy in the passive transfer EAE model. In this adoptive transfer EAE model, donor SJL (donor SJL) mice were immunized with CFA and PLP peptides. On day 10, total lymphocytes from draining LN (lymph nodes) were isolated and cultured for 4 days in vitro with PLP peptide, IL-23 (20 ng / ml) and anti-IFNg (10 g / ml). The expanded CD4 + T cells were purified and transplanted with naive recipient mice. Disease progression is monitored and has the following scores: 0, no disease; Loss of 0.5 tail tones; 1: limp tail; 2: limp tail + hind limb paresis; 2.5: one hind limb paralysis; 3: both hind limb paralysis; 3.5: forelimb weakness; 4: hind limb paralysis + unilateral forelimb paralysis. The mice are sacrificed when they reach a score of 4. * Is a sacrifice of a mouse.
Figure 18 shows that the anti-PD-L3 or VISTA antibody exhibits efficacy (reduces symptoms of arthritis) in a collagen-induced arthritis animal model.
Figure 19 shows that VISTA expressed on antigen presenting cells inhibits CD4 + T cell proliferation.
Figure 20 shows that anti-VSTA antibody inhibits tumor growth in mice transplanted with MB49 tumor cells.
Figure 21 shows the antitumor effect of VISTA mAb in four different mouse anti-tumor models.
Figure 22 shows the effect of the VISTA mAb on the efficacy of the CD40 / TLR agonist vaccine.
Figure 23 shows VISTA expression in CNS cells.
Figure 24 shows the effect of VISTA on the fate and function of T cells in the EAE model.

Before describing the present invention in more detail, the following definitions are provided.

As used herein, the term "immune cell" includes hematopoietic origin and cells that act as an immune response. Immune cells include lymphocytes such as B cells and T cells; Natural killer cells; And myeloid cells including monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, the term "T cell" includes CD4 + T cells and CD8 + T cells. In addition, the term T cell includes both T helper 1 type T cells and T helper 2 type T cells.

The term "antigen presenting cell" refers to a cell that expresses not only specialized antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells) but also other antigen presenting cells (e.g. keratinocytes, endothelial cells endothelial cells, astrocytes, fibroblasts, and oligodendrocytes.

The term "antigen" herein refers to an antigen whose modulation of the immune response to it may be therapeutically desirable. In the case of a favored enhanced immune response against a particular target antigen, the antigen includes, by way of example, but not limited to, infectious disease antigens from which a protective immune response can be induced. For example, the antigens from HIV under consideration are proteins gag, env, pol, tat, rev, nef, reverse transcriptase and other HIV components. In addition, E6 and E7 proteins from human papilloma virus are under consideration. In addition, EBNA1 antigens from herpes simplex virus are being considered. Other viral antigens under consideration include hepatitis viral antigens such as the S, M, and L proteins of the hepatitis B virus, the pre-S antigen of the hepatitis B virus, and other hepatitis, Hepatitis virus components such as Hepatitis A, B, and C, hepatitis C virus RNA; Influenza viral antigens such as hemagglutinin, neuraminidase, nucleoprotein, M2, and other influenza virus components; Measles viral antigens such as measles virus fusion proteins and other measles virus antigen components; Rubella viral antigens such as the proteins El and E2, and other luvealavirus antigen components; Rotaviral antigens such as VF7sc and other rotavirus antigen components; Cytomegaloviral antigens such as envelope glycoprotein B and other cytomegalovirus antigenic components; Respiratory syncytial viral antigens such as components of RSV fusion proteins, M2 proteins and other respiratory virus antigens; Immediate herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex virus antigenic components; varicella zoster viral antigens such as gpl, gpll, and other target herzin virus antigenic components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other components of the Japanese encephalitis virus antigen; Rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies virus antigenic components; West Nile virus prM and E proteins; And Ebola envelope protein. For additional examples of viral antigens, see Fundamental Virology, Second Edition, eds. Knipe, D.M., and Howley P.M. (Lippincott Williams & Wilkins, New York, 2001) for additional examples of viral antigens. It also contains bacterial antigens. Bacterial antigens that may be used in the compositions and methods of the invention include, but are not limited to, pertussis toxin, filamentous hemagglutinin, pertactin, FI2, FIM3, adenyl Pertussis bacterial antigens such as adenylate cyclase and other pertussis bacterial antigen components; Diptheria bacterial antigens such as diptheria toxin or toxoid and other diphtheria bacterial antigen components; Tetanus bacterial antigens such as components of tetanus toxins or toxoids and other tetanus antigens; Streptococcal bacterial antigens such as M protein and other streptococcal bacterial antigen components; Staphylococcal bacterial antigens such as IsdA, IsdB, SdrD, and SdrE; Gram-negative bacilli bacterial antigens such as lipopolysaccharides, flagellin, and other Gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), a 30 kDa major secretory protein and other mycobacterial antigen components; Helicobacter pylori bacterial antigen component; Pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza virus antigenic components; Anthrax bacterial antigens such as anthrax protective antigen, anthrax lethal factor, and other antlust bacterial antigen components; F1 and V proteins from Yersinia pestis; Rickettsiae bacterial antigens, such as romps and other rickettsia bacteria bacterial antigen components. It also includes any other bacteria, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens along with the bacterial antigens disclosed herein. Examples of protozoa and other parasitic antigens include, but are not limited to: merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, , Plasmodium falciparum antigens such as gametocyte / gamete surface antigens, blood-stage antigen pf 1 55 RESA and other malaria antigen components; Toxoplasma antigens such as SAG-1, p30 and other toxoplasma antigen components; Schistosomae antigens such as glutathione-S-transferase, paramyosin, and other members of the schizophrenia antigens; Leishmania such as leishmania major and other Richemian antigen components such as gp63 and trypanosoma cruzi antigen such as 75-77 kDa antigen, 556 kDa antigen and other typhanosome antigen components antigens). Examples of fungal antigens include, but are not limited to, Candida species, Aspergillus species, Blastomyces species, Histoplasma species, Coccidiodomycosis species, Malassezia furfur and other species, Exophiala werneckii and other species, Piedraia hortai and other species, Trichosporum beigelii and other species, For example, microsporum species, Trichophyton species, Epidermophyton species, Sporothrix schenckii and other species, Fonsecaea pedrosoi and other species , Wangiella dermatitidis and other species, Pseudallescheria boydii and other species, Madurella sp. Cyano (Madurella grisea) and other types, separation tank crispus types including (Rhizopus species), abscisic dia type (Absidia species), and non-cor type (Mucor species), but This allows not limited. Examples of prion disease antigens include PrP, beta-amyloid, and other prion-related proteins.

Another area of desirable enhanced immunogenicity for non-infectious agents as well as the above-mentioned infectious and parasitic agents is in the field of dysproliferative diseases and includes, but is not limited to, cancer Cells expressing cancer antigens are removed from the body. Tumor antigens and methods of the present invention that may be used in the compositions include prostate specific antigen (PSA), breast cancer, bladder cancer, ovarian cancer, testicular cancer, melanoma, telomerase; Multidrug resistance proteins such as P-glycoprotein; MAGE-1, alpha fetoprotein, carcinoembryonic antigen, mutant p53, papillomavirus antigens, gangliosides or carbohydrates of other melanoma or other tumor cells But are not limited to, carbohydrate-containing components. It is contemplated by the present invention that antigens from any kind of tumor can be used in the compositions and methods described herein. The antigen may be an immunogenic substance isolated from cancer cells such as cancer cells or membrane proteins. Survivin and endosomal rearrangement enzymes include the general antigen and the MAGE system of testicular cancer antigen. Antigens that may be used in the methods of the present invention that appear to be associated with autoimmunity and induce resistance include myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, and the CI1 collagen protein of Rheumatoid arthritis.

The antigen may be part of an infectious agent such as, for example, HZV-1, EBV, HBV, influenza virus, SARS virus, poxviruses, malaria or HSV. But are not limited to, for example, those in which a vaccine is required to harvest strong T cell mediated immunity (via dendritic cells).

The term "tumor" refers to at least one cell or cell in the form of tissue neoformation, usually in the form of overgrowth of endogenous tissue, specifically spontaneous, autonomous, and irreversible, , Which is usually associated with a loss of specific cell and tissue function. Such cells or cell masses are not effectively inhibited by themselves or by the control mechanisms of host organisms in relation to growth, examples being melanoma or carcinoma. Tumor antigens include not only the antigen present in or in the malignant cell itself, but also the antigen present in the stromal supporting the tissue of the tumor, including endothelial cells and other vascular components.

As used herein, an "immune response" includes T cell-mediated and / or B-cell immune responses that are effected by modulation of T cell co-stimulation. Exemplary immune responses include the activation of B cell responses (e.g., antibody production), T cell responses (e.g., cytokine production and cellular cytotoxicity) and cytokine response cells, such as macrophages do. As used herein, the term " down-regulation "of an immune response includes reduction in one or more immune responses, while the term" up-regulation " . It will be appreciated that upregulation of any one type of immune response may result in corresponding downregulation in another type of immune response. For example, upregulation of the production of certain cytokines (e.g., IL-10) may result in down regulation of the cellular immune response.

The term " co-stimulatory receptor ", as used herein, includes receptors that deliver a co-stimulatory signal to immune cells, such as CD28 or ICOS. The term " inhibitory receptor ", as used herein, includes receptors that deliver a voice signal to immune cells.

The term "co-stimulates ", as used herein, with respect to activated immune cells refers to a second, non-active, receptor-mediated signal (" co- stimulation signal ") of a co- stimulatory molecule, . For example, a co-stimulatory signal may result in cytokine secretion, for example, in a T cell that has received a T cell-receptor-mediated signal. Immune cells that receive a cell receptor-mediated signal are referred to herein as "activated immune cells, " for example, through an activating receptor.

Inhibitory signals delivered by inhibitory receptors may be useful when the co-stimulatory receptor (CD28 or ICOS) is not present in immune cells and thus is not a function of competition between inhibitory receptors and co-stimulatory receptors in binding of co-stimulatory molecules (Fallarino et al. (1998) J. Exp. Med. 188: 205). Delivery of inhibitory signals to immune cells may result in immune cell unresponsiveness, anergy, or programmed cell death. Preferably, the delivery of the inhibitory signal operates through a mechanism not associated with apoptosis.

As used herein, "apoptosis" includes cell planar apoptosis that can be characterized using techniques known in the art. Apoptosis can be characterized by chromosomal condensation, for example, the most pronounced in cell shrinkage, mambrane blebbing, and cell fragmentation. In addition, cells undergoing apoptosis exhibit a characteristic pattern of DNA cleavage in the internucleosome.

As used herein, an " autoimmune disease "is a disease or disorder caused by attacking and / or causing a co-segregate or manifestation of an individual's tissue. Examples of autoimmune diseases or disorders are arthritis [acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis Osteoarthritis, arthritis chronica progrediente, deformans arthritis, polyarthritis chronica primaria, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, Rheumatoid arthritis such as rheumatoid arthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin disease, plaque psoriasis, gutatte psoriasis, pustular psoriasis, Psoriasis such as psoriasis of the nails, contact dermatitis, chronic contact dermatitis, allergy Dermatitis including dermatitis, allergic contact dermatitis, herpetiformis dermatitis, and dermatitis including atopic dermatitis, X-linked and IgM syndrome, chronic allergic urticaria, and chronic autoimmune urticaria. , Urticaria such as chronic idiopathic urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic Sclerosis, systemic sclerosis, spino-optical MS, primary progressive MS, PPMS), and multiple sclerosis (MS) such as relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, dissemination Inflammatory bowel disease (IBD) such as, for example, Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative < RTI ID = 0.0 > The present invention relates to a method for the treatment of ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema (erythema) The primary sclerosing cholangitis (episcleritis) is classified into adult or respiratory distress syndrome including adult or acute respiratory distress syndrome (ARDS), meningitis, uvea Inflammation of the whole or a part of the body, iritis, choroiditis, autoimmune hematological disorder, rheumatoid spondylitis, acute hearing loss, anaphylaxis and allergies and atopy Inflammatory diseases such as IgE-mediated diseases such as rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, With or without nephrotic syndrome, such as uveitis, such as posterior uveitis, or autoimmune uveitis, chronic or acute glomerulonephritis such as primary GN (primary GN), or glomerulonephritis with or without nephrotic syndrome. GN), immuno-mediated GN, membranous GN, membranous nephropathy, idiopathic membranous nephropathy or idiopathic membranous GN, Renal Brain including type I and II, , Eczema including asthma bronchiale, bronchial asthma (including bronchial asthma), bronchial asthma (bronchial asthma), asthma, bronchitis, bronchial asthma and autoimmune asthma, diseases associated with T cell infiltration and chronic inflammatory reactions, chronic pulmonary inflammatory disease, myocarditis, leukocyte adhesion deficiency, systemic inflammation, Systemic lupus erythematosus (SLE) or systemic lupus erythematosus such as skin SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, Neuronal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus [nephritis, cerebritis, pediatric, non-renal, extra-renal (Type I) diabetes mellitus (including type 2 diabetes mellitus), which includes pediatric insulin-dependent diabetes mellitus (IDDM), including diabetes mellitus, discoid, alopecia, ), Adult-onset diabetes (type II), autoimmune diabetes, and idiopathic diabetes insipidus. Granulomatosis, Wegener's disease, including immune responses related to acute and delayed hypersensitivity mediated by T lymphocytes and cytokines, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, granulomatosis, agranulocytosis, vasculitis (including macular vasculitis (including polymyalgia rheumatica and Takayasu's arteritis), Kawasaki disease, polyarteritis nodosa, Systemic necrotizing vasculitis, Churg-Strauss vasculitis or syndrome (CSS), as well as for the treatment and / or prophylaxis of diabetic neuropathic pain, vasculopathy, vasculopathy, vasculitis, microscopic polyarteritis, CNS arthritis, necrosis, Vasculitides, including temporal arteritis, including < RTI ID = 0.0 > , Aplastic anemia, autoimmune aplastic anemia, coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or autoimmune hemolytic anemia (AIHA) Anemia perniciosa, Addison's disease, pure red cell anemia aplasia (PRCA), factor VHI deficiency, hemophilia A, autoimmune neutropenia, autoimmune hemolytic anemia, (autoimmune neutropenia), pancytopenia, leukopenia, diseases associated with leukocyte diapedesis, CNS inflammatory disorders, septicemia, trauma, hemorrhage. Antibody-mediated disease, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease (Bechet's or Behcet's disease, s disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, Pemphigoids such as pemphigoid bullous and skin pemphigoid, pemphigus [pemphigus vulgaris, pemphigus foliaceus (pemphigus foliaceus), pemphigus mucosal pseudogoid pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, temporal myelitis neuromyelitis optica, polyneuropathies, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, thrombotic platelets Thrombotic thrombocytopenic purpura; Thrombocytopenia (e. G., Developed by patients with myocardial infarction), including autoimmune or immuno-mediated thrombocytopenia, such as idiopathic thrombocytopenia (ITP), including chronic or acute ITP, Hypothyroidism including hypothyroidism such as primary hypothyroidism and autoimmune thyroiditis, autoimmune endocrine disease, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); Or polyglandular syndromes such as subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, autoimmune polyhedrin syndrome (or polyglandular endocrinopathy syndromes) paraneoplastic syndromes including neurotraumatic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man syndrome or stiff-man syndrome ), Encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia such as thymoma-associated myasthenia gravis gravis, cerebellar degeneration, Neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, Chronic hepatitis, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, Bronchiolitis obliteran versus NSIP, Guillain-Barre syndrome, Berger's disease, IgA nephropathy, idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, primary biliary cirrhosis biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, disease, celiac sprue, gluten enteropathy, refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis, ALS; Lou Gehrig's disease, coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus syndrome (eg, polychondritis) such as myoclonus syndrome (OMS), refractory or recurrent polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis including cancerous lymphocytosis, monoclonal B cell lymphopathy (eg, monoclonal gammopathy of undetermined significance (MGUS)), positive monoclonal gammopathy and undetermined significance ), Peripheral neuropathy, paraneoplastic syndrome, epilepsy, migraine, arrhythmia, muscular dysfunction, such as deafness, blindness, periodic paralysis, and channelopathies such as CNS channelopathy, autism, inflammatory myopathy, focal segmental glomerulosclerosis, Endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologicai disorder, fibromyalgia, multiple endocrine disorders, multiple sclerosis, multiple sclerosis, multiple sclerosis, (such as autoimmune demyelinating diseases) such as Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, autoimmune demyelinating diseases, demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcino sis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl, and telangiectasia), male and female infertility, mixed connective tissue disease, Chagas ' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, , Bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, allergic alveolar osteomalacia and fibrotic fungal infection, And are useful in the treatment of diseases such as alveolitis, interstitial lung disease, transfusion disease, leprosy, malaria, leishmaniasis, kypanosomiasis, asperia, schistosomiasis, ascariasis, aspergillosis, Sampter ' s syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, Fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, (E. G., Erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, chronic cystitis, diabetic sexual cystitis, irritable bowel syndrome, iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, Human immunodeficiency virus (HIV) HIV infection, echovirus infection, cardiomyopathy, Alzheimer ' s disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, streptococcal infections, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of nephritis, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, Chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic conjunctivitis, idiopathic nephritic syndrome And ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, and inflammatory diseases. The present invention relates to a method for the treatment and prophylaxis of idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, Bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis, , Autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic enteritis enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, (Eg, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of inflammatory bowel disease, including ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, osteoporosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, Infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo o), SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (SCID) AIDS), parasitic diseases such as Leschmania, toxic-shock syndrome, food poisoning, disease associated with T cell infiltration, leukocyte-adhesion deficiency, cytokines and T cells An antibody-antibody complex-mediated disease, antiglomerular basement membrane disease, allergic diseases, allergic diseases, allergy-related diseases, allergic diseases, The patient was diagnosed with allergic neuritis, autoimmune polyendocrinopathies, ovarian inflammation, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic disease rheumatic diseases, mixed connective tissue diseases, nephrotic syndrome, insulitis, polyendocrine failure, Peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, (S), dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non- Eosinophilia such as sinusitis, acute or chronic sinusitis, ethmoids, frontal, maxillary or sphenoid sinusitis, eosinophilia, pulmonary infiltration eosinophilia), eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilia Granulomas including chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or eosinophil, anaphylactic reactions including eosinophilia, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucosal candidiasis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, ), Autoimmune disorders associated with collagen diseases, rheumatism, neurological diseases, ischemic reperfusion disorders, A disease associated with a decrease in response, vascular degeneration, antigensis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and vascularization, Glomerulonephritides, reperfusion injury, reperfusion injury of cardiac muscle or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis, or other central pancreatitis Inflammatory diseases of the nervous system, ocular and orbital inflammatory diseases, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, Pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic giant-artery disorder, but are not limited to, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

"Cancer" and "cancerous " refer to or refer to a physical disease of a mammal that is characterized by generally uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of the cancer include squamous cell cancer, lung cancer (small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, (Including squamous carcinoma of the lung), peritoneal cancer, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma Cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, , Thyroid cancer, hepatocarcinoma (hepatic carcinoma), and head Neck cancer (head and neck cancer) variety, as well as B- cell lymphoma (B-cell lymphoma) of [longitude / follicle protection car kansong lymphoma (low grade / follicular non-Hodgkin's lymphoma; NHL); Small lymphocytic (SL) NHL; Intermediate grade / follicular NHL; Intermediate grade diffuse NHL; High grade immunoblastic NHL; High grade lymphoblastic NHL; High grade small non-cleaved cell NHL; Bulky disease (NHL); Mantle cell lymphoma; AIDS-related lymphoma; And Waldenstrom ' s Macroglobulinemia); Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Hairy cell leukemia; Chronic myeloblastic leukemia; Multiple myeloma and post-transplant lymphoproliferative disorder (PTLD).

The expression "allergic disease" refers to a disease associated with an allergic reaction. More specifically, "allergic disease" is defined as a disease in which allergens are identified, in which there is a close correlation between exposure to the allergen and the onset of pathological changes, Lt; / RTI > Here, the immunological mechanism means that leukocytes exhibit an immune response to allergen stimulation. Examples of allergen include mite allergen and pollen allergen. Representative allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis, and pollen and insect allergies. Allergic diathesis is a genetic factor that can be inherited from a child in an allergic parent. In addition, family allergic diseases are called atopic diseases, and the causative genetic factor is atopic specific. "Atopic dermatitis" is a generic term for diseases associated with apotheosis, particularly dermatitis. Preferred examples include selected allergic diseases from the group consisting of eczema, allergic rhinitis, hay fever, urticaria, and food allergies. Allergenic diseases include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria, hives and food allergies, and other atopic diseases.

"Asthma" means a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased respiratory responsiveness to inhaled agents. Asthma is often not only associated with atopic or allergic symptoms.

The expression "inflammatory disease or inflammatory disease" as used herein includes chronic or wet inflammatory diseases, including diseases or disorders selected from the group comprising: rheumatic diseases (rheumatoid arthritis, osteoarthritis, psoriatic arthritis Including, but not limited to, spondyloarthropathies (ankylosing spondylitis, reactive arthritis, Reiter's syndrome), crystalline (including but not limited to, arthropathies (including, but not limited to, gout, pseudogout, calcium pyrophosphate deposition disease), Lyme disease, rheumatic polymyalgia rheumatica; Including, but not limited to, connective tissue diseases (systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, Sjogren's syndrome) ]; But are not limited to, vasculitides (polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome); Inflammatory diseases including the consequences of trauma or ischaemia; Sarcoidosis; Atherosclerotic vascular disease, atherosclerosis and vascular occlusive disease [atherosclerotic heart disease, ischaemic heart disease, myocardial infarction, stroke Vascular diseases including, but not limited to, peripheral vascular disease, and vascular stent restenosis; Ocular diseases, including uveitis, corneal disease, iritis, iridocyclitis, and cataracts.

Cancers which can be treated by the present invention include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, But is not limited thereto. More specific examples of the cancer include bladder cancer, ovarian cancer, melanoma, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of lung, squamous cell cancer of lung), peritoneal cancer, hepatocellular carcinoma, Colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, ovarian cancer, ovarian cancer, hepatoma, bladder cancer, hepatoma, (B-cell lymphoma), as well as various types of hepatocellular carcinoma, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and head and neck cancer B-cell lymphoma [low grade / follicular non-Hodgkin's lymphoma (NHL)); Small lymphocytic (SL) NHL; Intermediate grade / follicular NHL; Intermediate grade diffuse NHL; High grade immunoblastic NHL; High grade lymphoblastic NHL; High grade small non-cleaved cell NHL; Bulky disease (NHL); Mantle cell lymphoma; AIDS-related lymphoma; And Waldenstrom ' s Macroglobulinemia); Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Hairy cell leukemia; Chronic myeloblastic leukemia; Multiple myeloma and post-transplant lymphoproliferative disorder (PTLD)], as well as phakomatoses, edema (such as those associated with brain tumors), and Meigs syndrome < / RTI > syndrome). Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, NHL, renal cell cancer, metastatic adenocarcinoma, liver cancer, pancreatic cancer, soft tissue Is selected from the group consisting of soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. In an exemplary embodiment, the cancer is an early-progressive (metastatic) bladder cancer, an ovarian cancer or a melanoma. In another embodiment, the cancer is a colorectal cancer. The cancer diseases that can be treated by the present invention include metastatic cancer wherein expression of VISTA by myeloid derived suppressor cells (MDSCs) is inhibited by antitumor and anti- invasive immune response. The method of the invention is particularly suitable for the treatment of vascular tumors.

The present invention also relates to a method for the treatment and / or prophylaxis of cancer, in combination with chemotherapy or radiotherapy or other biological therapies, for the treatment and prophylaxis of cancer, i. E. VISTA expression by bone marrow- Lt; / RTI > is suitable for individuals who inhibit the efficacy or biological efficacy of chemotherapy or radiation therapy. A chemotherapeutic agent exhibiting certain anticancer activity may be used according to the present invention. Preferably, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids vinca alkaloids, epipodopyyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination, complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressants, adrenocorticosteroids, progestins, estrogens, antiestrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone ≪ / RTI > gonadotropin-releasing hormone analog. More preferably, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), Ieucovorin (LV), irenotecan, oxaliplatin, capecitabine, Paclitaxel, and doxetaxel. ≪ / RTI > Two or more chemotherapeutic agents may be used in the cocktail being injected with the administration of the anti-VEGF antibody. One preferred concomitant chemotherapy is fluorouracil-based, which includes 5-FU and one or more other chemotherapeutic agents. Appropriate therapeutic doses of concomitant chemotherapy are well known in the art and are described, for example, in Saltz et al. (1999) Proc ASCO 18: 233a and Douillard et al. (2000) Lancet 355: 1041-7. Such biological therapies are potentially potent as cytokines, growth factor antagonists and agonists, hormones and anti-cytokine antibodies as well as PD-L1, PD-L2, CTLA-4 and PD-L1, PD- It may be an immune potentiator.

Depending on the form of the PD-L3 or VISTA molecule that binds to the receptor, the signal may be transmitted (e. G., In a multivalent form of PD-L3 or VISTA causing cross- Or by soluble forms of PD-L3 or VISTA binding to an Fc receptor in an antigen presenting cell) or competing with, for example, the activated form of the PD-L3 or VISTA molecule for binding to the receptor (For example, a soluble monovalent form of PD-L3 or VISTA molecule or a PD-L3 or PD-L3 molecule modified to not bind to an Fc receptor in an antigen presenting cell using methods known in the art) By dissolution of VISTA]. However, there are examples where the soluble molecule may be stimulatory. The effect of the various modulators can be readily described using a general screening test described herein.

As used herein, the term "activated receptor" includes an antigen, a complex antigen (e. G. In the context of an MHC molecule), or an immune cell receptor that binds to an antibody. Such activation receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors and Fc receptors.

For example, T cell receptors are present in T cells and are associated with CD3 molecules. T cell receptors are stimulated by antigens (as well as by polyclonal T cell activators) in the context of MHC molecules. T cell activation through TCR is associated with many changes, such as protein phosphorylation, membrane lipid changes, ionic flux, cyclic nuleotide alteration, transcriptional changes in RNA, protein synthesis Changes, and cell volume changes.

As used herein, the term "B cell receptor" (BCR) refers to a complex between membrane Ig (mIg) found in B cells and other membrane-passing polypeptides (eg, Ig alpha and Ig beta) . The signaling function of mIg is caused by the cross-linking of receptor molecules by oligomeric or multimeric antigens. In addition, B cells can be activated by anti-immunoglobulin antibodies. After BCR activation, many changes occur in B cells, including tyrosine phosphorylation.

The term "Fc receptor" (FcRs) includes cell surface receptors for the Fc portion of an immunoglobulin molecule (Igs). Fc receptors are found in many cells involved in the immune response. Human FcRs identified so far include IgG (Fc gamma.R), IgE (Fc Epsilon R1), IgA (Fc alpha R), and Polymer IgM / A (Fc. FcRs are found in the following cell types: Fc epsilon RI (mast cell), Fc epsilon. RII (many white blood cells), Fc alpha. R (neutrophil), and Fc mu (mu) alpha. (alpha) R (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9: 185-86). The widely studied Fc gamma Rs is at the heart of cellular immune defense and is responsible for stimulating the inflammatory and hydrolase mediator secretion associated with the onset of autoimmune disease (Unkeless, J. C (1988) Annu Rev. Immunol 6: 251-87). Since Fc gamma Rs provides a component of specific recognition that macrophages / monocytes, polymorphonuclear leukocytes and natural killer (NK) Fc gamma Rs are mediated by IgG, It provides an important link between leukocytes that secrete Ig. Human leukocytes have at least three different receptors for IgG: hFc gamma RI (found in macrophages / monocytes), hFc gamma RII (monocytes, neutrophils, eosinophils, platelets, possibly B cells, and K562 cell lines) , And Fc gamma (in NK cells, neutrophils, eosinophils, and macrophages).

For T cells, the transfer of co-stimulatory signals to T cells is associated with signaling pathways that are not inhibited by cyclosporin A. In addition, the co-stimulatory signal can induce cytokine (e.g., IL-2 and / or IL-10) secretion in the T cell and / or induce a no response to the antigen, induction of the innate, It is possible to prevent the induction of apoptosis,

As used herein, the term "inhibitory signal" refers to a signal transmitted through an inhibitory receptor molecule in an immune cell. Said signal antagonizes the signal through an activating receptor (e. G., Via a TCR, CD3 BCR, or Fc molecule) and may, for example, result in: formation of a second messenger; multiplication; Or a functional group in an immune cell, such as, for example, reduced macrophage, antibody production, or cellular cytotoxicity, or a mediator [eg, cytokine (eg, IL-2) and / Mediated] immune cell disorders; Or the development of Arnage.

As used herein, "unresponsiveness" includes stimulation, for example, refractivity of immune cells to stimulation via an activated receptor or cytokine. Non-response may occur, for example, due to exposure to immunosuppressants or high concentrations of antigens.

As used herein, the term "anergy" or "tolerance" includes a non-responsive state to an activating receptor-mediated stimulus. The unreacted state is generally antigen-specific and persists after exposure to a tolerizing antigen has ceased. For example, in T cells, anergy (as opposed to unreacted state) is characterized by a lack of cytokines, such as IL-2 formation. T cell neurons occur when T cells are exposed to an antigen and receive a primary signal (T cell receptor or CD3 mediated signal) in the absence of a secondary signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the secondary signal occurs in the presence of the co-stimulatory molecule) causes the formation of cytokines to fail, thus failing to proliferate. However, Annular T-cells can reach responses to unrelated antigens and may proliferate when cultured with cytokines (eg, IL-2). Also, for example, T cell secretion can be observed by lack of IL-2 formation by T lymphocytes by measuring using ELISA or using proliferation assays using indicator cell lines have. Alternatively, a receptor gene construct can be used. For example, Annuo T cells can express IL-2, which is induced by a heterologous promoter or by a multimer of API sequences that can be found in enhancers, under the control of the 5 'IL-2 gene. 2 gene transcription (Kang et al. (1992) Science 257: 1 134).

Control of the co-stimulatory signal results in regulation of the functional function of immune cells. Thus, the term "PD-L3 or VISTA activity" refers to the ability of a PD-L3 or VISTA polypeptide to bind its natural binding partner, the ability to modulate immune cell co-stimulatory or inhibitory signals, Including the ability to

Control of inhibitory signals in immune cells results in the regulation of proliferation and / or secretion of cytokines by immune cells.

As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence (which encodes a natural protein) occurring in nature.

As used herein, an "antisense" nucleic acid molecule is complementary to a "sense" nucleic acid that encodes a protein, e.g., complementary to a coding strand of a double-stranded cDNA molecule, It contains a nucleotide sequence complementary to the coding strand of the gene. Thus, the antisense nucleic acid molecule can hydrogen bond with the sense nucleic acid molecule.

As used herein, the term "coding regions" refers to a region of a nucleotide sequence that includes a codon translated into an amino acid residue, while a "noncoding region" (For example, the 5 'and 3' untranslated regions).

As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring another nucleic acid molecule to a connected site. One type of vector is a "plasmid ", which refers to a circular double stranded DNA loop to which additional DNA fragments can be ligated. Another type of vector is a viral vector, where additional DNA portions can be linked to a viral genome. Certain vectors can replicate themselves in the host cells into which they are introduced [e.g., bacterial vectors that have bacterial replication origin and episomal mammalian vectors]. Other vectors (e. G., Non-episomal mammalian vectors) are introduced into the host cell and then replicated in accordance with the host genome by integration into the genome of the host cell. In addition, certain vectors can manipulate gene expression. Wherein said vector refers to a "recombinant expression vector" or simply an "expression vector ". In general, the availability of expression vectors in recombinant DNA technology is often in the form of plasmids. In the context of the present invention, the terms "plasmid" and "vector" may be used interchangeably because the plasmid is the most categorical vector form. However, the invention also encompasses other forms of such expression vectors, such as viral vectors (e. G., Replication defective retroviruses, adenoviruses, and adeno-associated viruses) , Which has the same function.

As used herein, the term "host cell" refers to a nucleic acid molecule of the invention, e.g., a cell into which the recombinant expression vector of the invention is introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that the term refers not only to the cells of a particular individual but also to the progeny or potential progeny of the cell. Indeed, the progeny may not be identical to the parent cell, but are included within the scope of the term used herein, as certain modifications may occur in successive generations due to mutations or environmental effects.

 As used herein, a "transgenic animal" refers to a non-human animal, preferably a mammal, more preferably a mouse, wherein one or more cells of the animal are "transgenic" . The term "transgene" as used herein refers to the genome of an animal that is integrated into the genome of a cell developed in the transgenic animal and expresses the encoded gene product in one or more cells or tissues of the adult animal, Means the remaining exogenous DNA.

As used herein, "homologous recombinant animal" refers to a transgenic non-human animal, preferably a mammal, more preferably a species of a mouse, wherein the endogenous gene is an endogenous gene and the animal Cells, e. G., By homologous recombination between exogenous DNA molecules introduced into embryonic cells of the animal prior to development of the animal.

As used herein, the term "isolated protein" generally refers to a protein that is produced by recombinant DNA technology or is free of other proteins, cellular material and media when isolated from the cell, or when chemically synthesized, chemical precursors, or proteins that usually do not have other chemicals.

A "isolated " or" purified "protein, or a biologically active portion thereof, usually lacks cellular material or other contaminating proteins from cells or tissues from which the PD-L3 or VISTA protein is derived, Or other chemicals. The expression "cell material is usually absent" includes the preparation of a PD-L3 or VISTA protein separated from cell components of cells in which the protein is isolated or recombinantly produced. In some embodiments, the expression "no cellular material is usually present" is about 30% (by dry weight) or less of the non-PD-L3 or VISTA protein More preferably no more than about 10% of non-PD-L3 or VISTA protein, most preferably no more than about 5% of non-PD-L3 or VISTA protein RTI ID = 0.0 > PD-L3 < / RTI > or VISTA protein. When the PD-L3 or VISTA protein, or a biologically active portion thereof, is recombinantly produced, it is also usually preferred that the medium is absent. That is, the medium represents about 20% or less, more preferably about 10% or less, and most preferably about 5% or less of the protein preparation volume.

The expression "usually without chemical precursors or other chemicals" includes preparations of PD-L3 or VISTA proteins separated from chemical precursors or other chemicals associated with the synthesis of the protein. In some embodiments, the expression "there is usually no chemical precursor or other chemical" is expressed as about 30% (by dry weight) of the chemical precursor or non-PD-L3 or VISTA chemical, About 10% or less of the chemical precursor or non-PD-L3 or VISTA chemical, most preferably about 10% or less of the chemical precursor or non-PD-L3 or VISTA chemical, -PD-L3 or PD-L3 or VISTA protein having less than about 5% of the VISTA chemical.

The term "antibody ", as used herein, encompasses the entirety of an antigenic molecule as well as the" antigen-binding portion "(or simply" antigenic portion ") of the antibody. As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody having the ability to specifically bind to an antigen (eg, PD-L3 or VISTA). The antigen-binding function of the antibody was shown to be performed by a portion of the antibody full-lenth. Examples of binding fragments contained within the "antigen-binding portion" of an antibody include (i) a Fab fragment that is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F (ab ') 2 fragment of two fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 341: 544-546); And (vi) a separate complementarity determining region (CDR). Furthermore, even though the domains of VL and VH, which are domains of Fv fragments, are encoded by separate genes, they use a recombinant method to generate a single protein chain, in which the VL and VH sites are in pairs, (see, for example, Bird et al. (1988) Science 242: 423-44), which is known as single chain Fv (scFv) Proc Natl Acad Sci USA 85: 5879-5883 and Osbourn et al 1998 Nat Biotechnol 16: 778). In addition, the single chain antibody is included within the term "antigen-binding site" of the antibody. Some VH and VL sequences of a specific scFv may be linked to a human immunoglobulin constant region cDNA or genomic sequence to form an expression vector encoding a complete IgG molecule or other isotype. VH and VL can also be used in the formation of other fragments of Fab, Fc or immunoglobulin using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies have shown that VH and VL domains are expressed in a single polypeptide chain, but by using a very short linker to allow pairing between the two domains in the same chain, (For example, Holliger, P. et al. (1993) Proc Natl. Acad. Sci. USA), which allows the domain to generate two antigen- 90: 6444-6448; Poljak, RJ et al. (1994) Structure 2: 1 121-1 123).

In addition, the antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of said antibody or antibody portion with one or more other proteins or peptides. An example of such immunoadhesion molecules is the use of streptavidin central site to create tetrameric scFv molecules (Kipriyanov, SM et al. (1995) Hum. Antibodies Hybridomas 6: 93-101), and The use of cysteine residues, marker peptides and C-terminal polyhistidine tags to create bi- and biotinylated sc Fv molecules (Kipriyanov, SM et al. (1994) Mol Immunol. 31: 1047-1058). Antibody fragments, such as Fab and F (ab ') 2 fragments of whole antibodies, can be prepared from whole antibodies using common techniques, such as, for example, papain or pepsin digestion. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using the general recombinant DNA techniques described herein.

Antibodies can be polyclonal or monoclonal: xenogeneic, allogeneic, or syngeneic; Or a modified form thereof, for example, humanization, chimeric, and the like. Preferably, the antibodies of the invention specifically or highly specifically bind PD-L3 or VISTA molecules. The term "monoclonal antibody" and "monoclonal antibody composition ", as used herein, refers to a population of antibody molecules comprising only one species of antigen binding site capable of immunoreacting with a particular epitope of an antigen Quot; polyclonal antibody "and" polyclonal antibody composition "refer to a population of antibody molecules comprising multiple species of antigen binding sites capable of immunoreacting with a particular epitope of an antigen do. Monoclonal antibody compositions generally exhibit a single binding affinity for a particular antigen that undergoes an immune response.

The term "humanized antibody " as used herein includes antibodies made by non-human cells that have variable and constant regions that are modified to more closely mimic antibodies that can be made by human cells. For example, non-human antibody amino acid sequences are transformed into amino acid sequences found in human germline immunoglobulin sequences. The humanized antibodies of the invention can be used to detect, for example, amino acid residues that are not encoded by human intervening immunoglobulin sequences in CDRs (e.g., random or site-specific mutagenesis in vitro or in vivo Somatic mutation " within < / RTI > The term "humanized antibody ", as used herein, also includes antibodies that have been transplanted into human framework sequences with CDR sequences derived from other mammalian, e. G. .

As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificity (for example, a separate antibody that binds to PD-L3 or VISTA is usually referred to as PD-L3 Or no antibody that specifically binds to an antibody other than VISTA). In addition, isolated antibodies are often free of other cellular materials and / or chemicals.

The term " oligomerization domain "as used herein refers to a domain that promotes oligomalization when binding to the VISTA extracellular domain or a fragment thereof. The oligomerization domain may be further stabilized by additional disulfide bonds. Helix (self-associating .alpha. -Helics), such as leucine zipper. The domain is designed to coexist with vectorial folding beyond the membrane, a process thought to promote the folding of polypeptides into functional binding proteins in vivo. Examples thereof are known in the art and include coiled GCN4 and COMP as one example.

The above-mentioned .alpha -helical coiled coil is perhaps the oligomerization motif subunit found in the most prevalent proteins. Thus, the twisted spiral also satisfies a variety of different functions. For example, in several family transcriptional activators, short leucine zippers play a very important role in positioning DNA-binding sites in DNA (Ellenberger et al., 1992, Cell 71: 1223-1237 ). Also, twisted spirals are used to form oligomers of intermediate filament proteins. Also, twisted helical proteins appear to play an important role in both vesicle and viral membrane fusion (Skehel and Wiley, 1998, Cell 95: 871-874). In both cases, the hydrophobic sequence to be incorporated into the membrane to be melted is located at the same end of the rod-shaped complex. This molecular arrangement is considered to result in a close proximity to the membrane in order for the composite to be assembled for membrane fusion. The twisted spiral is often used to control oligomerization. This is found in many types of proteins, including, but not limited to, GCN4, a viral fusion peptide, a SNARE complex and a specific tRNA synthetase. Very long twisted spirals are found in proteins such as tropomyosin, intermediate fibers and spindle-pole-body components. Also, twisted spirals are associated with many .alpha.-helixes supercoiled around each other in a very ordered way involving in parallel or antiparallel directions. Although the dimer and trimer are most common, the helix can be from the same or different proteins. Twisted spirals can also be formed by the helix components that come together along with their hydrophobic seams. The hydrophobic boundary line surrounds each helix, and the helix is also wrapped around each other to form a super helix with a hydrophobic boundary line. This is a characteristic interdigitation of the side chain between neighboring helixes, also known as knobs-into-holes packing, defined by a twisted helical structure. The helix does not need to be oriented in the same direction for this kind of mutual coupling to occur, although the parallel conformation is more common. In trimer and pentamer antiparallel shapes are very rare, but more common in dimers between molecules, where two helixes are often connected by short loops. In the extracellular space, the heterotrimeric coiled-coil protein laminin plays an important role in the formation of basement memebrane. Another example is thrombospondins and cartilage oligomeric matrix protein (COMP), wherein three (tromospondin 1 and 2) or five (tromospondin 3, 4 and COMP) do. The molecule has a bouquet-like shape, and the cause of this oligomer structure is probably the multivalent interaction of the cell receptor and the C-terminal domain. Yeast transcriptional activator GCN4 is one of thirty identified eukaryotic proteins, including basic region leucine zipper (bZIP) DNA-binding motifs (Ellenberger et al., 1992, Cell 71: 1223-1237). The bZIP dimer is a pair of continuous alpha helices that form a parallel or twisted helix at their carboxyl-terminal 34 residues and gradually diverge toward their amino ends to pass through the major groove of the DNA binding site to be. Said another twisted-helical duplex contact is almost perpendicular to the DNA axis and forms a T-shaped complex. bZIP includes 4-3 7 repeats of hydrophobic and nonpolar residues to collect together in a parallel alpha-helix or twisted-helix (Ellenberger et al., 1992, Cell 71: 1223-1237). The stability of the dimer is shown by the density of leucine and nonpolar residues per side and the crystal structure of the GCN4 leucine zipper peptide at the a and d positions of 7 repeats (Ellenberger et al., 1992, Cell 71: 1223-1237), helix inter- and inter-spaced salt bridges. Another example is a subunit of Mr 52,000 homotrimer, CMP [matrilin-1], isolated from the bovine tracheal cartilage (Paulsson and Heinegard, 1981, Biochem J. 197 : 367-375), where each subunit consists of a vWFAl module, a single EGF domain, a vWFA2 module, and a twisted spiral domain spanning five 7 repeats (heptads) (Kiss et al., 1989 , J. Biol. Chem. 264: 8126-8134; Hauser and Paulsson, 1994, J. Biol. Chem. 269: 25747-25753). Electron microscopy of the purified CMP shows a bouquet-like trimer structure, wherein each subunit also forms an ellipse that is representative of a common point corresponding to a twisted helix (Hauser and Paulsson, 1994, J. Mol. Biol Chem 269: 25747-25753). The above-mentioned twisted helical domains in methirell-1 have been extensively studied. The trimer structure appears after complete reduction of interchain disulfide bonds under non-denaturing conditions (Hauser and Paulsson, 1994, J. Biol. Chem. 269: 25747-25753). Another example is the Cartilage Oligomeric Matrix Protein (COMP). COMP, the non-collagenous glycoprotein, was first identified in cartridges (Hedbom et al., 1992, J. Biol. Chem. 267: 6132-6136). The protein has four repeats of the N-terminal 7 repeats (EGF), the EGF-like domain (EF), the seven calcium-laden domains (T3), and the C- cc), a 524 kDa homopentamer of five subunits. According to this domain configuration, COMP belongs to the tromosphonidine system. Under the preferred hydrophobic residue 7 repeats (Heptad repeats, abcdefg), helices or a twisted-helical domain is formed at a and d (Cohen and Parry, 1994, Science 263: 488-489). Recently, the recombinant five-stranded coiled-coil domain of COMP (COMPcc) has been crystallized and its structure appears at a resolution of 0.2 nm (Malashkevich et al., 1996, Science 274 : 761-765).

The term "family" when referring to a polypeptide and nucleic acid molecule of the present invention refers to two or more polypeptides having a common structural domain or motif and having amino acid or nucleotide sequence homology as defined herein, or A nucleic acid molecule has an intent to mean. The system members can occur naturally or non-naturally and can be from one of the same or different species. For example, the system may comprise the first polypeptide of human origin and other, distinct human origin polypeptides, or may comprise homologues of non-human origin, such as monkey polypeptides. In addition, members of the system may have general functional characteristics.

For example, the systems of PD-L3 and VISTA polypeptides of the invention preferably comprise at least one "signal peptide domain ". As used herein, "signal sequence" or "signal peptide" includes peptides comprising 15 or more amino acids that occur at the N-terminus of the secreted and membrane bound polypeptides and that contain many hydrophobic amino acid residues. For example, the signal sequence comprises at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, even more preferably about 19 amino acid residues Wherein the signal sequence comprises at least about 35-65% of a hydrophobic amino acid residue (e. G., Valine, leucine, isoleucine or phenylalanine) 38-50%, more preferably about 40-45%. The "signal sequence" is also referred to in the art as a "signal peptide" and functions to direct a polypeptide comprising the sequence to a lipid bilayer, and is cleaved into secreted and membrane bound polypeptide forms. As described below, the signal sequence was identified in the amino acid sequence of the present human PD-L3 or VISTA (native human PD-L3 or VISTA), and also in the mouse PD-L3 or VISTA (native mouse PD-L3 OR VISTA) Amino acid residues.

In another embodiment of the invention, the PD-L3 or VISTA polypeptides of the invention have been identified based on the presence of a "transmembrane domain ". As used herein, "transmembrane domain" includes amino acid sequences of about 15 amino acids in length, which pass through a plasma membrane. More preferably, the transmembrane domain comprises at least about 20, 25, 30, 35, 40, or 45 amino acid residues and penetrates the plasma membrane. The transmembrane domain has many hydrophobic residues and generally has an alpha-helical structure. In a preferred embodiment, the amino acid of the transmembrane domain is at least 50%, 60%, 70%, 80%, 90%, 95% or more hydrophobic, such as leucine, isoleucine, tyrosine or tryptophan tryptophan). For example, the transmembrane domain is described by Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19: 235-263, which is incorporated herein by reference. The transmembrane domain of PD-L3 is identified herein (see, e.g., Fig. 1).

In another embodiment, a PD-L3 or VISTA molecule of the invention has been identified based on the presence of an "IgC domain" and the absence of an "IgC domain" in the polypeptide or its corresponding nucleic acid molecule. The IgV and IgC domains used herein are recognized in the art as Ig superfamilies. These domains are consistent with structural units with a distinct folding pattern called Ig folds. Ig folds contain sandwiches of two beta sheet structures, each of which is usually, but not all, inversely parallel beta-rays of 5-10 amino acids with conserved disulfide bonds between the two panel structures (beta strand). The IgC domains of Ig, TCR, and MHC molecules share the same kind of sequence pattern and are called C1 sets in the Ig chain. Other IgC domains fall into different sets. In addition, IgV domains also share sequence patterns and are called V-set domains. The IgV domain is longer than the C-domain and forms additional pairs of beta-rays. The amino acid residues of the human and mouse PD-L3 or VISTA polypetide, which are composed of IgV domains, are shown in FIG. The presence of the IgV domain appears to be essential for the binding of PD-L3 or VISTA or its natural binding partner.

In yet another embodiment, the PD-L3 or VISTA molecules of the invention have been identified based on the presence of an "extracellular domain" in the polypeptide or the corresponding nucleic acid molecule. As used herein, the term "extracellular domain" refers to an N-terminal amino acid extending as a tail from the cell surface. The extracellular domain of the present invention comprises an IgV domain and may comprise a signal peptide domain (see Figure 1).

In yet another embodiment, the PD-L3 or VISTA molecules of the invention have been identified based on the presence of a "cytoplasmic domain" in the folepredide or its corresponding nucleic acid molecule. As used herein, the term "cytoplasmic domain" refers to the C-terminus of the amino acid extending as a tail in the cytoplasm of a cell, which is expected to constitute the cytoplasmic domain.

In a preferred embodiment, PD-L3 or VISTA of the present invention comprises at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

A separate polypeptide, preferably a PD-L3 or VISTA polypeptide of the invention has an amino acid sequence that is sufficiently identical to the amino acid sequence of SEQ ID NO: 2 or 4, or 5, or has an amino acid sequence that is sufficiently similar to SEQ ID NO: 1 or 3 or a fragment or complement thereof Lt; RTI ID = 0.0 > nucleotide < / RTI > The term "sufficiently identical" as used herein refers to a first amino acid or nucleotide sequence comprising a sufficient or minimal number of amino acid residues or nucleotides identical or equivalent (e.g., having similar side chains) , A common structural domain or a first and a second amino acid or nucleotide sequence sharing motifs and / or common functional activity. For example, they share a common structural domain and have at least 30%, 40% or 50% homology, preferably 60% homology, more preferably 70% -80% homology in the amino acid sequence of the domain, , More preferably 90% -95% homology, and at least one and preferably two structural domains or motifs are defined herein to be sufficiently identical. Also, an amino acid or nucleotide sequence that shares at least 30%, 40% or 50%, preferably 60%, more preferably 70-80% or 90-95% homology and shares a common functional activity, Is defined to be the same.

"PD-L3 or VISTA activity", "biological activity of PD-L3 or VISTA" or "functional activity of PD-L3 or VISTA" used interchangeably herein refers to the activity of PD-L3 or VISTA- Polypeptide or nucleic acid molecule of PD-L3 or VISTA, as determined in accordance with the general techniques in vivo or in vitro at the PD-L3 or VISTA polypeptide binding partner. This activity involves modulating CD4 + and CD8 + T cell proliferation and cytokine production. In yet another embodiment, the PD-L3 or VISTA activity is direct activity, such as that associated with the PD-L3 or VISTA binding partner. As used herein, a "target molecule" or "binding partner" refers to a PD-L3 or VISTA polypeptide expressed in nature, i.e., a T cell, Or interacting with each other. Alternatively, PD-L3 or VISTA activity is an indirect activity, such as cell signaling activity mediated by PD-L3 or VISTA polypeptides. For example, the PD-L3 or VISTA polypeptides and PD-L3 or VISTA agonists or antagonists of the invention may have one or more of the following activities: (1) the biological activity of PD-L3 or VISTA, ) Inhibits or promotes the proliferation of CD4 + and CD8 + T cells, (2) inhibits or promotes cytokine production, (3) inhibits T cell responses during cognate interactions between T cells and bone marrow derived APCs (4) inhibit early TCR activation and arrest cell division with minimal effect on apoptosis, thereby inhibiting CD4 + T cells from acting as a negative regulatory ligand (5) inhibits or promotes antigen-specific T cell activation during covalent interactions between APCs and T cells, and / or (6) inhibits T cell-mediated immune response ≪ / RTI > (7) regulates the activation of immune cells, such as T lymphocytes, and (8) regulates the inflammatory immune response of immune responses, such as organisms, such as mice or human organisms.

Thus, another embodiment of the invention features isolated PD-L3 or VISTA proteins and polypeptides that modulate one or more PD-L3 or VISTA activities. Such a polypeptide will comprise a PD-L3 or VISTA polypeptide having one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and preferably PD-L3 or VISTA active.

Additionally preferred PD-L3 or VISTA polypeptides may have at least one extracellular domain, and one or more signal peptide domains, IgV domains, transmembrane domains, and cytoplasmic domains, and preferably under certain hybridization conditions, Or a nucleic acid molecule having a nucleotide sequence that hybridizes with a nucleic acid molecule comprising the complement of the nucleotide sequence of SEQ ID NO: 1 or 3. Exemplary, nucleotide and amino acid sequences of isolated human and mouse PD-L3 or VISTA cDNA and predicted amino acid sequences of human PD-L3 or VISTA polypeptides are included in the sequences listed herein.

Human VISTA or PD-L3 or VISTA has been identified as being upregulated in T cell transcription profile screening. Characterization of the same 930 bp gene product obtained from the murine CD4 + T-cell cDNA library confirmed these sizes and sequences. The silico-sequence and structural analysis indicate that it is a type I membrane penetrating protein of 309 amino acids after maturation. Its extracellular domain contains a single extracellular Ig-V domain of 136 amino acids, which is linked to a 23-amino acid stalk region, a 21-residue transmembrane region, and a 97-amino acid cytoplasmic domain. The cytoplasmic tail of VISTA does not contain any signal domain. BLAST sequence searches using VISTA Ig-V domomain identified B7 PD-L1 as the evolutionarily closest related protein with a borderline significant e-value score. The structural basic sequence alignment with B7 family members PD-L1, PD-L2, B7-H3 and B7-H4 highlighted several amino acids conserved systematically in all Ig-V domain proteins.

Various aspects of the invention are set forth in the following detailed description.

I. PD - L3  or VISTA  The isolated nucleic acid molecule

One aspect of the present invention is directed to a method for identifying an isolated nucleic acid molecule, PD-L3 or VISTA-encoding nucleic acid molecule (e.g., PD-L3 or VISTA mRNA) encoding a PD-L3 or VISTA polypeptide or a biologically active portion thereof As well as nucleic acid fragments sufficient for use as hybridization probes and as PCR primers for amplification of PD-L3 or VISTA nucleic acid molecules or for many years. As used herein, the term "nucleic acid molecule" refers to a DNA molecule (e.g., cDNA or genomic DNA) and an RNA molecule (e.g., mRNA) and a nucleotide analog ≪ / RTI > The nucleic acid molecule may be a single strand or a double strand, but preferably a double stranded DNA.

The term "isolated nucleic acid molecule" includes nucleic acid molecules separated from other nucleic acid molecules present in the natural origin of the nucleic acid molecule. For example, for genomic DNA, the term "isolated " includes nucleic acid molecules in which the genomic DNA is isolated from a naturally associated chromosome. Preferably, the "isolated" nucleic acid molecule is a liberated sequence (ie, a sequence located at the 5 'and 3' ends of the nucleic acid molecule) naturally occurring in the genomic DNA of the nucleic acid from which the nucleic acid is derived. For example, in various embodiments, the isolated PD-L3 or VISTA nuclear molecule has a nucleotide sequence that is naturally located on the nucleic acid molecule side in the genomic DNA of the cell from which the nucleic acid molecule is derived is about 5 kb, 4 kb, 3 kb , 2 kb, 1 kb, 0.5 kb or 0.1 kb or less. In addition, "isolated" nucleic acid molecules, such as cDNA molecules, can be other cellular material or media that have been liberated, usually produced by recombinant techniques, or can be chemical precursors or other chemicals that are liberated when chemically synthesized have.

Nucleic acid molecules of the invention, for example, nucleic acid molecules having SEQ ID NOS: 1, 3, or portions thereof, may be separated using general molecular biology techniques and the sequence information provided herein. A PD-L3 or VISTA nucleic acid molecule can be hybridized using conventional hybridization and cloning techniques (for example, S. mefaus, Ambrook, J. et al. Molecular < RTI ID = Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Also, a nucleic acid molecule comprising SEQ ID NO: 1, 3 or an ortholog or a whole or part of a mutation can comprise a synthetic oligonucleotide primer designed based on the sequence of SEQ ID NO: 1, 2, 3, 4 or 5 Or by polymerase chain reaction (PCR).

The nucleic acid molecule of the present invention can be amplified according to conventional PCR amplification techniques using cDNA, mRNA, or genomic DNA as a template, using appropriate oligonucleotide primers. The highly amplified nucleic acid molecule can be cloned into an appropriate vector and characterized by DNA sequencing. Oligonucleotides along the PD-L3 nucleotide sequence can also be prepared by conventional synthetic techniques, for example, using an automated DNA synthesizer.

In a preferred embodiment, the isolated PD-L3 or VISTA encoding the nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a fragment thereof. In yet another embodiment, the nucleic acid molecule of the invention comprises a complementary nucleic acid molecule of the nucleotide sequence shown in SEQ ID NO: 1 or 3 or any portion of the nucleotide. The complementary nucleic acid molecule of the nucleotide sequence shown in SEQ ID NO: 1 or 3 is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3 and can be hybridized with the nucleotide sequence shown in SEQ ID NO: Thereby forming a stable duplex.

In another preferred embodiment, the isolated nucleic acid of the invention has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 85%, at least about 80% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical nucleotide sequences.

Also, a nucleic acid molecule of the invention may comprise a probe that encodes only a portion of the nucleic acid sequence of SEQ ID NO: 1 or 3, for example a portion of PD-L3 or a VISTA polypeptide, such as a PD-L3 or a biologically active portion of a VISTA polypeptide Or fragments that can be used as primers or fragments. The nucleotide sequence determined from the cloning of the human PD-L2 gene enables the generation of probes and primers designed for use in identifying and / or cloning other PD-L3 family members as well as PD-L3 or VISTA homologs from other species do. The probe / primer generally comprises highly purified oligonucleotides. The oligonucleotides generally comprise, under certain conditions, a sense sequence of SEQ ID NO: 1 or 3; At least about 12 or 15, preferably at least about 20 or 25, more preferably at least about 30, at least about 35, or at least about 30, , 40, 45, 50, 55, 60, 65, or 75 contiguous nucleotides of the nucleotide sequence.

In certain embodiments, the nucleic acid molecules of the invention can be in the range of about 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500- 550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950 or more nucleotides and, in certain hybridization conditions, 1 or 3, or a complementary nucleic acid molecule thereof. In yet another embodiment, the nucleic acid molecule of the invention has a length of about 880-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150 or more nucleotides and, in certain hybridization conditions, A nucleotide sequence that hybridizes to a nucleic acid molecule of SEQ ID NO: 1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule of the invention has a length of 50-100, 100-150, 150-200, 200-250, 250-300 or more nucleotides and, in certain hybridization conditions, SEQ ID NO: 1 or 3, or a nucleotide sequence that hybridizes with a nucleic acid molecule comprising a complementary sequence thereof. In yet another embodiment, the nucleic acid molecules of the invention can be present in the range of about 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500 1-550, 550-600, 600-650, 650-700, 700-750, 750-800, 850-900, 900-950 or more nucleotides in length, with the coding region of SEQ ID NO: 1 or 3, A nucleic acid molecule comprising at least about 15 (i.e., 15 contiguous) nucleotide sequences and including a nucleotide sequence as shown in SEQ ID NO: 1 or 3, or a complement thereof, Lt; RTI ID = 0.0 > nucleotides < / RTI >

Probes based on PD-L3 or VISTA nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous polypeptide. In a preferred embodiment, the probe comprises a label group associated therewith, for example yth or an enzyme cavity surface marker group may be a radioactive isotope, a fluorescent compound, an enzyme cofactor. The probe may detect PD-L3 or VISTA mRNA levels, for example, by measuring the level of PD-L3 or VISTA-encoded nucleic acid in a cell sample from an individual, or detecting a PD-L3 or VISTA mRNA level, The PD-L3 or VISTA polypeptide can be used as part of a diagnostic test kit that detects a cell or tissue that is mis-expressed, such as determining whether the PD-L3 or VISTA polypeptide is deleted or not.

A nucleic acid that encodes a " biologically active portion of a PD-L3 or VISTA polypeptide "has PD-L3 or VISTA biological activity (e.g., ability to bind its natural binding partner and / or modulate immune cell activity) (E. G., By recombinant expression in vitro) by separating portions of the nucleotide sequence of SEQ ID NO: 1 or 3 that encode a polypeptide having the PD-L3 or VISTA polypeptide, and PD Lt; RTI ID = 0.0 > of-L3 < / RTI > or VISTA polypeptides.

The present invention also encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 or 3 by a genetic code of degeneracy of genetic code and thus are encoded by the nucleotide sequence shown in SEQ ID NO: Lt; RTI ID = 0.0 > PD-L3 < / RTI > or VISTA polypeptide. In yet another embodiment, the isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, or 5.

DNA sequence polymorphisms that result in changes in the amino acid sequence of PD-L3 or VISTA polypeptides by those skilled in the art, as well as the PD-L3 or VISTA nucleotide sequences shown in SEQ ID NOS: 1 and 3, , ≪ / RTI > a human population). Within the PD-L3 or VISTA genes, the genetic polymorphism can exist among individuals within a population due to natural allelic variation. As used herein, the term "gene" or "recombinant gene" includes an open reading frame encoding PD-L3 or VISTA polypeptides, preferably mammalian PD-L3 or VISTA polypeptides, Quot; means a nucleic acid molecule that may comprise non-coding regulatory sequences and an intron.

Allelic variants of human or murine PD-L3 or VISTA include both functional and non-functional PD-L3 or VISTA polypeptides. Functional allelic variants are human or mouse PD-L3 or VISTA polypeptides that retain the ability to bind to native PD-L3 or VISTA binding partners and / or to modulate CD4 + and CD8 + T cell proliferation and cytokine production and lipofugal activation Lt; / RTI > sequence variation of naturally occurring amino acids. Functional allelic variations generally involve conservative substitutions of one or more amino acids of SEQ ID NO: 2, 4 or 5, or substitution, deletion or insertion of non-essential residues at non-critical sites of the polypeptide.

Non-functional allelic variants may occur naturally in human or mouse PD-L3 or VISTA polypeptides that bind to native PD-L3 or VISTA binding partners and / or are not capable of modulating any activity of PD-L3 or VISTA described herein This is an amino acid sequence variation. A non-functional allelic variation is generally a non conservative substitution, deletion or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 2, 4 or 5, or an important residue or important region of the polypeptide, Insertion, or deletion in the IgV domain.

The present invention also provides non-human, non-murine orthologs of human or murine PD-L3 or VISTA polypeptides. The heterologous forms of PD-L3 or VISTA polypeptides in humans or mice are isolated from nonhuman, non-murine organisms and have the same binding affinity and / or specificity as the human or murine PD-L3 or VISTA polypeptides described herein, Or lymphocyte activation-modulating activity and the proliferation and cytokine production of CD4 + and CD8 + T cells. The heterologous homologue of human or murine PD-L3 polypeptide can be readily identified because it contains the amino acid sequence which is generally identical to SEQ ID NO: 2, 4 or 5.

Also included in the scope of the present invention are nucleic acid molecules encoding different PD-L3 or VISTA family members, and thus nucleic acids having different nucleotides from the PD-L3 or VISTA sequence of SEQ ID NO: 1 or 3. For example, another PD-L3 or VISTA cDNA can be identified based on the nucleotide sequence of mouse or human PD-L3 or VISTA. Also included in the scope of the invention are nucleic acid molecules encoding PD-L3 or VISTA polypeptides from different species, and thus nucleic acid molecules having a nucleotide sequence that differs from the PD-L3 or VISTA sequence of SEQ ID NO: 1 or 3. For example, monkey PD-L3 or VISTA cDNA can be identified based on the nucleotide sequence of mouse or human PD-L3 or VISTA.

The nucleic acid molecule corresponding to the natural allelic variation and homology of the PD-L3 or VISTA cDNA of the present invention is a hybridization probe according to a general hybridization technique under specific hybridization conditions, and the PD -L2 nucleic acids, or portions thereof. ≪ RTI ID = 0.0 > Naturally occurring allelic variants and homologous nucleic acid molecules of PD-L3 or VISTA cDNA of the present invention can be isolated by mapping to the same chromosome or locus as the PD-L3 or VISTA gene .

Thus, in another embodiment, the isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and, under certain conditions, has a coding region of the nucleotide sequence of SEQ ID NO: 1 or 3 ≪ / RTI > In yet another embodiment, the nucleic acid molecule is at least 700, 750, 800, 850, 880-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150 or more nucleotides in length do.

As used herein, the term "hybridize under specific conditions" is intended to describe conditions for hybridization and washing, in which nucleotide sequences substantially identical to each other or homologous to one another remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other are maintained hybridized to one another. Such specific conditions are known to those skilled in the art and are found in Current Protocols in Molecular Biology, Ausubel et al., Eds., John Wiley & Sons, Inc (1995), sections 2, 4, Additional specific conditions are described in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9, and 11. Preferred, non-limiting examples of specific hybridization conditions include, but are not limited to, 4x or 6x sodium chloride / sodium citrate (SSC), 65-70C (占 폚) (or 4x SSC and 50% formamide (formamide), hybridization at 42-50 degrees C), preferably additionally followed by washing at 1 X SSC, 65-70 degrees C, and non-limiting examples of specific conditions include 6 times SSC, 45 degrees C , Followed by one or more washes at 0.2X SSC, 1 @ SDS, 65 DEG C. Preferred, non-limiting examples of very stringent hybridization conditions include hybridization at 1X SSC, hybridization at 65-70C (or 1X SSC and 50% formamide, hybridization at 42-50C) 0.3 times SSC, 65-70 degrees C, and one or more washes. Preferred, non-limiting examples of less stringent specific hybridization conditions include hybridization at 4- or 6-fold SSC, 50-60 ° C (or 6-fold SSC and 50% formamide, hybridization at 40-45 ° C) 2 x SSC, 50-60 C, and one or more washes. The intermediate range of the above-mentioned numerical values, for example, 65-70 degrees C or 42-50 degrees C is included by the present invention. SSPE (1X SSPE is 0.1 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) in hybridization and wash buffer was performed with SSC [1X SSC in 0.15M NaCl and 15 mM sodium citrate )]; After hybridization is complete, washing is carried out for 15 minutes each. The hybridization temperature for a hybrid expected to be less than 50 base pairs in length should be 5-10 degrees C below the melting temperature Tm of the hybrid, . (Degrees C.) = 2 (number of A + T bases) +4 (number of G + C bases) for hybrids having a length of 18 base pairs or less. (Degrees C.) = 81.5 + 16.6 (loglO [Na +]) + 0.41 (% G + C) - (600 / N) for hybrids between 18 and 49 bases in length, where N [Na + 1] is the concentration of sodium ion in the hybridization buffer ([Na. +] = 0.165 M for 1X SSC). It will also be appreciated by those skilled in the art that additional reagents may be added to the hybridization and / or wash buffer to reduce nonspecific hybridization of nucleic acid molecules to membranes, such as nitrocellulose or nylon membranes (For example, BSA or salmon or herring sperm carrier DNA), a detergent (e.g., SDS), a chelating agent (e.g., EDTA), a chelating agent ≪ / RTI > Ficoll, PVP, and the like. In particular, when using nylon membranes, preferred, non-limiting examples of specific hybridization conditions include hybridization at 0.25-0.5M NaH2PO4, 7% SDS, at about 65 ° C, followed by 0.02M NaH2PO4, 1% SDS, Lt; RTI ID = 0.0 > and / or < / RTI > For example, Church and Gilbert (1984) Proc. Nad. Acad. Sci. USA 81: 1991-1995 (or 0.2-fold SSC, 1% SDS).

Preferably, the isolated nucleic acid molecule of the present invention is hybridized with the sequence of SEQ ID NO: 1 or 3 or its corresponding naturally occurring nucleic acid molecule under certain conditions. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule (i. E., Encoding a versatile polypeptide) having a nucleotide sequence occurring in nature.

As well as the naturally occurring allelic variation of the PD-L3 or VISTA sequence that may be present in a population, those skilled in the art will appreciate that variations can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1 or 3 and thus the PD-L3 or VISTA polypeptide Will result in a change in the amino acid sequence of the encoded PD-L3 or VISTA polypeptide, without a change in the functional ability of the PD-L3 or VISTA polypeptide. For example, substitution of nucleotides resulting in amino acid substitutions in a "non-essential" amino acid residue can be made in the sequence of SEQ ID NO: 1 or 3. A "non-essential" amino acid residue is a residue that can be modified without a change in biological activity from the wild type (eg, SEQ ID NO: 2, 4 or 5) of PD-L3 or VISTA, Is essential for biological activity. For example, amino acid residues conserved among the PD-L3 or VISTA polypeptides of the invention, such as those present in the extracellular domain, are predicted to be unacceptable, especially for modification. In addition, additional amino acid residues conserved between the PD-L3 or VISTA polypeptides of the invention and other members of the PD-L3 or VISTA family can not be tolerated against modification.

Thus, another aspect of the invention includes nucleic acid molecules encoding PD-L3 or VISTA polypeptides that include changes in amino acid residues that are not essential for activity. The PD-L3 or VISTA polypeptide differs in amino acids from SEQ ID NO: 2, 4 or 5 but retains biological activity. In some embodiments, the isolated nuclear eye comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide has at least 71%, 75%, 80%, 85%, 90% identity to SEQ ID NO: 2, , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence.

An isolated nucleic acid molecule encoding a PD-L3 or VISTA polypeptide identical to the polypeptide of SEQ ID NO: 2, 4 or 5 is produced by introducing into the nucleotide sequence of SEQ ID NO: 1 or 3 the substitution, addition or deletion of one or more nucleotides , Whereby one or more amino acid substitutions, additions or deletions can be introduced into the encoded polypeptide. Mutations, such as site-directed mutagenesis and PCR-mediated mutagenesis, may be introduced by SEQ ID NO: 1 or 3 by common techniques. Preferably, the conserved amino acid substitutions can be made in one or more non-essential amino acid residues. A " conservative amino acid substitution "is one in which the amino acid residue is substituted with an amino acid residue having a similar side chain. Systems of amino acid residues having similar side chains are defined in the art. Such systems include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) Serine, threonine, tyrosine, cysteine), uncharged polar side chains (for example, asparagine, glutamine, serine, threonine, For example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , Beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine ), Phenylalanine, tryptophan, and histidine]. Thus, non-essential amino acid residues predicted in PD-L3 or VISTA polypeptides are replaced by another amino acid residue from the same side chain system windy. Alternatively, in another embodiment, the mutation may be randomly introduced by all or part of the PD-L3 or VISTA coding sequence, for example by saturation mutagenesis, and the resulting mutation may be PD-L3 or VISTA Can be screened with the above activity for mutant identification with biological activity. In the mutation formation of the following column number 1 or 3, the encoded polypeptide can be recombinantly expressed and the activity of the polypeptide can be determined.

In a preferred embodiment, the mutant PD-L3 or VISTA polypeptide binds to and / or controls natural PD-L3 or VISTA, modulates intracellular or intracellular signaling, modulates the activation of T lymphocytes, And / or the ability to modulate the immune response of an organism.

Another aspect of the invention includes isolated nucleic acid molecules encoding PD-L3 or VISTA, or PD-L3 or VISTA fusion proteins. The nucleic acid molecule comprising a first nucleotide encoding a PD-L3 or VISTA protein, polypeptide or peptide operatively linked to a second nucleotide sequence encoding at least a PD-L3 or VISTA protein, polypeptide or peptide is produced by a general recombinant DNA technique . ≪ / RTI >

In addition to nucleic acid molecules encoding the PD-L3 or VISTA polypeptides, another aspect of the invention includes isolated nucleic acid molecules that are antisense thereto. An "antisense" nucleic acid molecule includes a nucleotide sequence complementary to a "sense" nucleic acid molecule encoding a polypeptide, for example, complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence. Thus, the antisense nucleic acid can hydrogen bond with the sense nucleic acid. The antisense nucleic acid may be complementary to the entire PD-L3 or VISTA cipher or only a portion thereof. In some embodiments, the antisense nucleic acid molecule is antisense to the "coding site" of the coding strand of the nucleotide sequence encoding PD-L3 or VISTA. The term "coding region" refers to a portion of a nucleotide sequence that includes a codon that is translated into an amino acid residue. In another embodiment, the antisense nucleic acid molecule is antisense to the "unencrypted site" of the coding strand of the nucleotide sequence encoding PD-L. The term "non-coding region" means a 5 ' and 3 ' sequence (also referred to as a 5 ' and 3 ' non-translated region) on the side of an encoding site that is not translated into an amino acid. In the cipher sequence encoding human or mouse PD-L3 or VISTA disclosed herein, the antisense nucleic acid of the present invention is designed according to the base pairing rules of Watson and Crick. The antisense nucleic acid molecule may be complementary to the entire coding region of PD-L3 or VISTA mRNA, but is preferably an oligonucleotide that is antisense to only a portion of the coding or non-coding region of the PD-L3 OR VISTA mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation initiation site of PD-L3 OR VISTA mRNA. For example, antisense oligonucleotides are about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acid molecules of the present invention can be made using chemical synthesis and enzymatic linkage reactions known in the art. For example, an antisense nucleic acid molecule (e. G., An antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides, or can be chemically synthesized to increase the biological stability of the molecule or between the antisense and sense nucleic acids Various modified nucleotides, such as phosphorothioate derivatives and acridine substituted nucleotides, can be used to increase the physical stability of the formed duplexes. An example of a modified nucleotide that can be used to generate an antisense nucleic acid is 5-fluorouracil, 5-bromouracil, 5-chloroouracil, 5-iodouracil ), Hyxoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- Carboxymethylaminomethyl-2-thiouridine, 5-carboxyrnethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6 - N-isopentenyladenine, 1-memylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine 2-methyladenine, 2-methylguanine, 3-methylcytosine, ne, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5- 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v) But are not limited to, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil- Uracil-5-oxyacetic acid (v), 5-methyl-2 3-amino-3-N-2-carboxypropyl) uracil], (3-amino-3-N-2-carboxypropyl) acp3) w, and 2,6-diaminopurine. Or the antisense nucleic acid can be produced biologically by using an expression vector with a nucleic acid subcloned into the antisense flavor (i.e., the RNA transcribed from the inserted nucleic acid will be the antisense orientation for the target target nucleic acid, Which will be described in more detail below.

The antisense nucleic acid molecules of the present invention are generally administered to a subject or produced in situ so that they are hybridized with intracellular mRNA and / or genomic DNA encoding PD-L3 or VISTA polypeptides, thereby inducing expression of the polypeptide Inhibiting, e.g., transcription and / or translation. The hybridization may be carried out by a universal nucleotide complement to form a stable duplex, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex, a specificity in the deep groove of a double helix Can be formed through mutual mutual coupling. An example of the administration route of the antisense nucleic acid molecule of the present invention includes direct injection at the tissue site. Alternatively, the antisense nucleic acid molecule can be administered systemically after being injected into the target selected cells. For example, for systemic administration, the antisense molecule is modified and specifically binds to a receptor or antigen expressed on the surface of the selected cell. For example, by linking the antisense nucleic acid molecule with a peptide or antibody that binds to a cell surface receptor or antigen. In addition, antisense nucleic acid molecules can be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecule, a vector construct in which the antisense molecule is located under the control of a strong pol or pol III promoter is preferred.

In another embodiment, the antisense nucleic acid molecule of the invention is an alpha.-Anomeric nucleic acid molecule. .Alpha.-anomeric nucleic acid molecules form specific double helical hybrids with complementary RNA, where strands, which are the opposite of the usual .beta.-units, (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). The antisense nucleic acid may also be a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215: 327-330).

In another embodiment, the antisense nucleic acid molecule of the invention is a ribozyme. A ribozyme is a catalytic RNA molecule with ribonuclease activity capable of cleaving single-stranded nucleic acids, such as mRNA, with complementary sites. Thus, ribozymes (eg, described in Haseloff and Gerlach (1988) Nature 334: 585-591) can be used to catalytically cleave PD-L3 or VISTA mRNA transcripts , The translation of PD-L3 or VISTA mRNA can be inhibited. A ribozyme having specificity for PD-L3 or VISTA-encoding nucleic acid can be designed based on the nucleotide sequence of PD-L3 or VISTA cDNA disclosed herein (i.e., SEQ ID NO: 1 or 3). For example, a derivative of Tetrahymena L-19 IVS RNA (Tetrahymena L-19 IVS RNA) is made in such a manner that the nucleotide sequence of the active site is complementary to a nucleotide sequence that can be cleaved in PD-L3 or VISTA- Can be. For example, Cech et al., U.S. Pat. Pat. No. 4,987,071 and Cech et al., U.S. Pat. Pat. No. 5,116,742. Alternatively, PD-L3 or VISTA mRNA can be used to screen for catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel, D. and Szostak, J. W. (1993) Science 261: 1411 -1418.

Alternatively, the PD-L3 or VISTA gene expression may be regulated by a regulatory region of PD-L3 or VISTA (e.g., PD-L3 (SEQ ID NO: 3)) to form a triple helix that prevents transcription of the PD- Or the VISTA promoter and / or the enhancer). Generally, Helene, C (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C et al. (1992) Ann. N.Y. Acad. Sci. 660: 27-36; and Maher, L. J. (1992) Bioessays (12): 807-15.

In another embodiment, the PD-L3 or VISTA nucleic acid molecules of the present invention may be used in combination with a base moiety, a sugar moiety, or a peptide moiety to improve stability, hybridization or solubility of the molecule, It can be modified in a phosphate backbone. For example, the deoxyribose phosphate backbone of the nucleic acid molecule can be modified to generate a peptide nucleic acid (Hyrup, B. and Nielsen, PE (1996) Bioorg. Med. Chem. l): 5-23). As used herein, the term "peptide nucleic acid" or "PNAs" refers to nucleic acid mimics, such as DNA mimics, wherein the deoxyribose phosphate backbone is a pseudopeptide backbone , Leaving only four natural nucleobases. The neutral backbone of PNAs has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described by Hyrup and Nielsen (1996) and Perry-O'Keefe et al. (1996) Proc Natl. Acad. Sci. USA 93: 14670-675. ≪ Desc / Clms Page number 11 >

PNAs of PD-L3 or VISTA nucleic acid molecules can be used for therapeutic and diagnostic uses. For example, PNAS scans can be used as antisense or anti-gene agents for sequence-specific regulation of gene expression, for example, by inducing transcription or translation stoppage or by inhibiting replication. PNAs of PD-L3 or VISTA nucleic acid molecules can also be used in the analysis of single base pair mutations in the gene (e.g., by PNA-directed PCR clamping); Can be used as "artificial restriction enzymes" (eg, SI nucleases (Hyrup and Nielsen (1996)) or as probes or primers for DNA sequencing or hybridization when used in combination with other enzymes (Hyrup and Nielsen (1996); Perry-O'Keefe et al. (1996) supra).

In yet another embodiment, PNAs of PD-L3 or VISTA bind lipid affinity or other helper groups to PNA (for example, to enhance their stability or uptake) DNA chimeras, or by using liposomes or other drug delivery techniques known in the art. For example, PNA-DNA chimeras of PD-L3 or VISTA nucleic acid molecules can be generated by fusing the merits of PNA and DNA. The chimera allows DNA recognition enzymes (e.g., RNAse H and DNA polymerase) to interact with the DNA moiety, while the PNA moiety can provide high binding affinity and specificity. The PNA-DNA chimeras can be linked using a linker of appropriate length, selected in terms of base stacking, number of bonds between nucleotide bases and orientation (Hyrup and Nielsen (1996)). . The synthesis of PNA-DNA chimeras is described by Hyrup and Nielsen (1996) and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, the DNA chain can be modified using standard phosphoramidite coupling chemistry and modified nucleoside analogs such as 5 '- (4-methoxytrityl) amino-5 Can be synthesized on a solid support using 5'- (4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, and the bridge between PNA and DNA 5 ' (Mag, M. et al. (1989) Nucleic Acids Res. 17: 5973-88). The PNA monomer is then coupled in a stepwise fashion to form chimeric molecules with the 5 'PNA portion and the 3' DNA portion (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized using the 5 'DNA portion and the 3' PNA portion (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

In another embodiment, the oligonucleotides may be used in conjunction with other added groups, such as peptides (e. G., To target host cell receptors in vivo or in cell membranes (Letsinger et al. (1989) Proc Natl. Acad. USA 86: 6553-6556; Lemaitre et al. (1987) Proc Natl Acad Sci USA 84: 648-652; PCT Application No. WO 88/09810) or blood-brain barrier (See, for example, PCT Application No. WO 89/10134). Oligonucleotides may also be used in hybridization-triggered cleavage agents (Krol et al. (1988) Biotechniques 6 : 958-976) or an intercalating agent (see Zon (1988) Pharm. Res. 5: 539-549). Finally, the oligonucleotide can be modified with another molecule [ For example, peptides, hybridization triggered cross-linking agents, transposers rt agent or a hybridization-induced cleavage agent).

Alternatively, expression characteristics of an endogenous PD-L3 or VISTA gene in a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into a stable cell line or a genome of a cloned microorganism, Lt; RTI ID = 0.0 > PD-L3 < / RTI > or VISTA gene. For example, an endogenous PD-L3 or VISTA gene that is normally "transcriptionally silent" in normal cell lines or microorganisms, i.e., PD-L3 or VISTA genes are not expressed normally or only at very low levels, Can be activated by inserting regulatory elements that can promote the expression of gene products normally expressed in cell lines or microorganisms. Alternatively, an endogenous PD-L3 or VISTA gene that is globally silent can be activated by injecting a promiscuous regulatory element that works across several cell types.

A heterologous regulatory element may be inserted into a stable cell line or a cloned microorganism, which is well known in the art and described in Chappel, U.S. Pat. Pat. No. 5,272,071; PCT Application No. < / RTI > filed May 16, 1991; Can be operatively linked to endogenous PD-L3 or VISTA genes by using techniques such as targeted homologous recombination, described in WO 91/06667.

II . Isolated PD - L3  or VISTA  Polypeptides, and anti- PD - L3  or VISTA  Antibody

One aspect of the invention includes PD-L3 or VISTA polypeptides, and biologically active portions thereof, polypeptide fragments suitable for use as immunogens for raising anti-PD-L3 or VISTA antibodies. In some embodiments, the native PD-L3 or VISTA polypeptide can be isolated from cells or tissue sources by appropriate purification schemes using conventional protein purification techniques. In yet another embodiment, PD-L3 or VISTA polypeptides are produced by recombinant DNA technology. As an alternative to recombinant expression, PD-L3 or VISTA proteins or polypeptides can be chemically synthesized using common peptide synthesis techniques.

A "isolated " or" purified "polypeptide, or a biologically active site thereof, is usually found in the absence or presence of cellular material or other contaminating proteins from cells or tissues from which the PD-L3 or VISTA protein is derived, Or other chemicals. The expression "cell material is usually absent" includes the preparation of a PD-L3 or VISTA protein separated from cell components of cells in which the protein is isolated or recombinantly produced. In some embodiments, the expression "no cellular material is usually present" is about 30% (by dry weight) or less of the non-PD-L3 or VISTA protein More preferably no more than about 10% of non-PD-L3 or VISTA protein, most preferably no more than about 5% of non-PD-L3 or VISTA protein RTI ID = 0.0 > PD-L3 < / RTI > or VISTA protein. When the PD-L3 or VISTA protein, or a biologically active portion thereof, is recombinantly produced, it is also usually preferred that the medium is absent. That is, the medium represents about 20% or less, more preferably about 10% or less, and most preferably 5% or less of the protein preparation volume.

The expression "usually without chemical precursors or other chemicals" includes preparations of PD-L3 or VISTA proteins separated from chemical precursors or other chemicals associated with the synthesis of the protein. In some embodiments, the expression "there is usually no chemical precursor or other chemical" is expressed as about 30% (by dry weight) of the chemical precursor or non-PD-L3 or VISTA chemical, About 10% or less of the chemical precursor or non-PD-L3 or VISTA chemical, most preferably about 10% or less of the chemical precursor or non-PD-L3 or VISTA chemical, -PD-L3 or PD-L3 or VISTA protein having less than about 5% of the VISTA chemical.

As used herein, a "biologically active site" of a PD-L3 or VISTA polypeptide refers to a mutual binding between a PD-L3 or VISTA molecule and a natural ligand of a non-PD-L3 or VISTA molecule, Or fragments of participating PD-L3 or VISTA polypeptides. The biologically active portion of the PD-L3 or VISTA polypeptide is substantially identical to, or substantially identical to, the amino acid sequence of the PD-L3 or VISTA polypeptide, such as, for example, the amino acid sequence shown in SEQ ID NO: Wherein the sequence comprises less than the full length of the PD-L3 or VISTA polypeptide and exhibits at least one activity of the PD-L3 or VISTA polypeptide. Generally, a biologically active site modulates (inhibits) at least one activity of a PD-L3 or VISTA polypeptide, e. G., CD4 + T cell proliferation to anti-CD3, the proliferation of associated CD4 + T cells in an antigen- Inhibition of the response, effect on expression of specific cytokines, and the like. The biologically active site of a PD-L3 or VISTA polypeptide can be, for example, a polypeptide of 25, 50, 75, 100, 125, 150, 175, 200, 225 or more amino acids in length. Biologically active sites of PD-L3 or VISTA polypeptides may be used as targets to develop PD-L3 or VISTA-mediated activities, e. G., Agents that modulate immune cell activation.

In some embodiments, the biologically active portion of the PD-L3 or VISTA polypeptide comprises at least a portion of an extracellular domain. A preferred biologically active site of a PD-L3 or VISTA polypeptide of the invention may comprise at least a portion of an extracellular domain (including, for example, IgV), and one or more of the following domains: a signal peptide domain, Domains, and cytoplasmic domains. In addition, other biologically active sites in which other regions of the polypeptide have been deleted can be produced by recombinant techniques, and one or more functional activities of native PD-L3 or VISTA polypetides can be assessed.

In a preferred embodiment, the PD-L3 or VISTA polypeptide has the amino acid sequence shown in SEQ ID NO: 2, 4, or 5. In yet another embodiment, the PD-L3 or VISTA polypeptide is substantially identical to SEQ ID NO: 2, 4, or 5 and retains the functional activity of the polypeptide of SEQ ID NO: 2, 4, or 5, The amino acid sequence differs by gene mutation.

In addition, the nucleic acid and polypeptide sequences of the present invention may be used as an "query sequence" to search for published databases to identify, for example, other system members or related sequences. The search is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 using the NBLAST and XBLAST programs (version 2.0). In order to obtain a nucleotide sequence having homology with the PD-L3 or VISTA nucleic acid molecule of the present invention, the BLAST nucleotide search can be performed within the NBLAST program, score = 100, wordlength = 12. BLAST protein searches can be performed within the XBLAST program, score = 100, wordlength = 3 to obtain nucleotide sequences that are homologous to PD-L3 or VISTA polypeptides of the present invention. For comparison purposes, Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402, Gapped BLAST can be used. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. You can refer to the Internet website for the National Center for Biotechnology Information.

The invention also provides PD-L3 or VISTA chimeric or fusion proteins. PD-L3 or VISTA "chimeric protein" or "fusion protein ", as used herein, includes a PD-L3 or VISTA polypeptide operatively linked to a non-PD-L3 or VISTA polypeptide. &Quot; PD-L3 or VISTA polypeptide "refers to a polypeptide having an amino acid sequence corresponding to a PD-L3 or VISTA molecule, while" non-PD-L3 or VISTA polypeptide "means a polypeptide having sufficient homology Quot; means a polypeptide having an amino acid sequence corresponding to a polypeptide that does not have the amino acid sequence of SEQ ID NO: 2, e.g., PD-L3 or VISTA polypeptide, and that is derived from the same or different organism. Within the PD-L3 or VISTA fusion protein, the PD-L3 or VISTA polypeptide may correspond to all or a portion of the PD-L3 or VISTA polypeptide. In a preferred embodiment, the PD-L3 or VISTA fusion protein comprises at least one portion of a biological PD-L3 or VISTA active site. In yet another embodiment, the PD-L3 or VISTA fusion protein comprises at least two domains of PD-L3 or VISTA polypeptides. Within the fusion protein, the term "operably linked" refers to the fusion of PD-L3 or VISTA polypeptides and non-PD-L3 or VISTA polypeptides to each other in-frame. A non-PD-L3 or VISTA polypeptide can be fused to the N-terminal or C-terminal of a PD-L3 or VISTA polypeptide and can be used to alter the solubility, binding affinity, stability, or valency of a PD-L3 or VISTA polypeptide Indicate with moiety

For example, in some embodiments, the PD-L3 or VISTA fusion protein is a GST-PD-L3 or VISTA fusion protein, wherein the PD-L3 or VISTA sequence is fused with the C-terminus of the GST sequence. The fusion protein may facilitate purification of the recombinant PD-L3 or VISTA. In another embodiment, the fusion protein is a PD-L3 or VISTA polypeptide comprising a heterologous signal sequence at the N-terminus. In certain host cells (e. G., Mammalian host cells), the expression and / or secretion of PD-L3 or VISTA may be increased through the use of heterologous signal sequences. In a preferred embodiment, the fusion protein is an Ig-PD-L3 or VISTA fusion protein, wherein the PD-L3 or VISTA sequence is fused to a portion of the Ig molecule. The Ig portion of the fusion protein may be an immunoglobulin constant region, such as the human C gamma 1 domain or the C gamma 4 domain (e.g., the hinge region of human IgC gamma 1 or human IgC gamma 4, CH2, and CH3 (See Capon et al., US Pat. Nos. 5, 1 16,964; 5,580,756; 5,844,095 and the like, incorporated herein by reference). The resultant fusion protein can change PD-L3 or VISTA solubility, binding affinity, stability and / or binding property (i.e., the number of binding sites per molecule) and improve the efficiency of protein purification.

In particular, the preferred PD-L3 or VISTA Ig fusion protein comprises an extracellular domain site associated with an immunoglobulin constant region (e.g., an Fc region) of PD-L3 or VISTA. Immunoglobulin constant regions may include genetic modifications that reduce or eliminate intrinsic functional activity in the immunoglobulin structure. For example, DNA encoding the extracellular portion of a PD-L3 or VISTA polypeptide can be obtained by introducing a DNA encoding the hinge region, CH2 and CH3 region of human IgG gamma 1 and / or IgG gamma 4 and a DNA encoding a position-directed mutation, 0.0 > WO 97/28267. ≪ / RTI > The PD-L3 or VISTA fusion protein of the present invention may be contained in a pharmaceutical composition and administered to an individual in vivo. The PD-L3 or VISTA fusion protein can be used to affect the bioavailability of the PD-L3 or VISTA binding partner. The use of PD-L3 or VISTA fusion proteins may be therapeutically useful for the treatment of diseases or intestines that may benefit from modulation of the immune response. In addition, the PD-L3 or VISTA-fusion protein of the present invention can be used to purify PD-L3 or VISTA-binding protein to produce an anti-PD-L3 or VISTA antibody in an alternative, Or as an immunogen to identify molecules that inhibit the interaction of VISTA and its natural binding partners.

Preferably, the PD-L3 or VISTA chimera or fusion protein of the invention is produced by common recombinant DNA techniques.

The invention also encompasses variants of PD-L3 or VISTA polypeptides that function as PD-L3 or VISTA agonists (mimetics) or as PD-L3 or VISTA antagonists. Variations of PD-L3 or VISTA polypeptides can be generated by PD-L3 or VISTA by mutagenesis, for example, discrete point mutation, or truncation of PD-L3 or VISTA polypeptides . Agonists of PD-L3 or VISTA polypeptides may have substantially the same or a subset of the biological activity of naturally-occurring forms of PD-L3 or VISTA polypeptides. Antagonists of PD-L3 or VISTA polypeptides may competitively compete for PD-L3 or VISTA-mediated activity of one or more of the naturally occurring forms of PD-L3 or VISTA polypeptide activity, for example PD-L3 or VISTA polypeptides Can be suppressed. Thus, a specific biological effect can be induced by treating a limited functional variation. In certain embodiments, treating variant individuals having a subset of the biological activity of the naturally occurring forms of the polypeptide has lower side effects than treating the subject in naturally occurring forms of PD-L3 or VISTA polypeptides.

In some embodiments, a variant of a PD-L3 or VISTA polypeptide that functions as a PD-L3 or VISTA agonist (mimetic) or as a PD-L3 or VISTA antagonist is administered to a subject for PD-L3 or VISTA polypeptide agonist or antagonist activity PD-L3 or VISTA polypeptides, for example, by screening combinatorial libraries of truncation mutants. In some embodiments, various libraries of PD-L3 or VISTA variants can be generated by combination mutagenesis at the nucleic acid level and are encoded by various gene libraries. A variety of libraries of PD-L3 or VISTA variants can be produced, for example, by enzymatically linking a mixture of synthetic oligonucleotides to a gene sequence, whereby a degenerate set of potential PD-L3 or VISTA sequences Can be expressed as individual polypeptides, or as sets of larger fusion proteins (e. G., Phage display), including sets of PD-L3 or VISTA sequences herein. There are a variety of methods that can produce a library of potential PD-L3 or VISTA variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerative gene sequences can be performed in an automatic DNA synthesizer, which is then linked to an appropriate expression vector. The use of a degenerate set of genes allows all of the sequences to encode a desirable set of potential PD-L3 or VISTA sequences in a mixture. Methods for synthesizing degenerative oligonucleotides are known in the art (Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477)

In addition, a fragment library of PD-L3 or VISTA polypeptide encoding sequences can be used to generate various populations of PD-L3 or VISTA fragments to screen for and select mutations of PD-L3 or VISTA polypeptides. In some embodiments, a library of coding sequence fragments can be generated by treating the double-stranded PCR fragment of the PD-L3 or VISTA coding sequence under conditions with a nuclease, wherein the conditions are such that nicking is about one molecule , Renaturation of the DNA from different cleaved products to denature double helix DNA and formation of double helix DNA that can contain sense / antisense pairs, and treatment with SI nuclease The single helix portion is removed from the reproduced duplex, and the resulting fragment is ligated to the expression vector. In this way, an expression library can be derived, which encodes N-terminal, C-terminal and internal fragments of various sizes of PD-L3 or VISTA polypeptides.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products with selected characteristics. This technique is adapted for rapid screening of gene libraries generated by combined mutagenesis of PD-L3 or VISTA polypeptides. The most widely used technique, which can have a high throughput analysis for large genomic library screening, involves cloning a gene library into a replicable expression vector, transforming the resulting vector library into appropriate cells And expressing said combination gene under conditions, wherein said condition promotes the isolation of a vector encoding a gene whose detection of the desired activity is to detect said product. Recursive ensemble mutagenesis (REM), which promotes the frequency of functional mutations in the library, can be used in conjunction with screening assays to identify PD-L3 or VISTA mutations (Arkin and Youvan (1992) Proc Natl. Acad Sci USA 89: 781 1-7815; Delagrave et al. (1993) Protein Eng 6 (3): 327-331).

PD-L3 or VISTA peptidomimetics as well as PD-L3 or VISTA polypeptides containing only naturally occurring amino acids are also provided. Peptide analogs are non-peptide drugs that have properties similar to those of the template peptide, and are generally used in the pharmaceutical industry. The types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger ) And Evans et al. (1987) J. Med. Chem 30: 1229 are incorporated herein by reference) and are sometimes developed for the purpose of computerized molecular modeling. Therapeutically useful peptides and structurally similar peptide mimetics can be used to produce the same therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to paradigm polypeptides (i. E., Polypeptides having biological or pharmaceutical activity), such as human or mouse PD-L3 or VISTA, but include linkages selected from the group consisting of: - CH2NH-, -CH2S-, -CH2-CH2-, -CH.dbd.CH- (cis and trans), -COCH2-, -CH2CH2-, - CH (OH) CH2-, and -CH2SO-, which are known in the art and are described by reference to the following references: Spatola, AF in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins Weinstein, B. , ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. I, Issue 3, "Peptide Backbone Modifications "; Morley, J. S. (1980) Trends. Pharm. Sci. pp. 463-468; Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14: 177-185 (-CH2NH-, CH2CH2-); Spatola, A. F. et al. (1986) Life. Sci. 38: 1243-1249 (-CH2-S); Hann, M. M. (1982) J. Chem. SoC Perkin. Trans. I 307-314 (-CH-CH-, cis and trans); Almquist, R. G. et al. (1980) J. Med. Chem. 23: 1392-1398 (-COCH2-); Jennings-White, C et al. (1982) Tetrahedron Lett. 23: 2533 (--COCH2 -); Szelke, M. et al., European Patent Application No. EP 45665 (1982) CA: 97: 39405 (~ CH (OH) CH2-); Holladay, M. W. et al. (1983) Tetrahedron. Lett. 24: 4401-4404 (-C (OH) CH2); and Hruby, V. J. (1982) Life Sci. 31: 189-199 (--CH2 S); Each of which is incorporated herein by reference. A particularly preferred non-peptide bond is -CH2NH. Such peptide mimetics may have the advantage of having significant advantages over native polypeptides, including, for example, the following: more economical production, higher chemical stability, enhanced pharmacological properties (e.g., half life, , Efficacy, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, and others. The label of the peptidomimetic can be detected either directly or indirectly by the quantitative structure-activity data and / or the on-interfering position in the peptidomimetic predicted by molecular modeling, ) (E. G., The amide group). ≪ / RTI > The non-interfering position is a position that does not generally make direct contact with the macromolecules to which the peptidomimetic binds, in order to produce a therapeutic effect. Derivatization (e. G., Labeling) of the peptidomimetic should not normally interfere with the desired biological or pharmacological activity of the peptidomimetic.

Systemic substitution of one or more amino acid sequences of the PD-L3 or VISTA amino acid sequence with the same class of D-amino acids (e.g., D-lysine at the position of L-lysine) produces a more stable peptide Lt; / RTI > In addition, limited peptides can be produced by methods known in the art, such as PD-L3 or VISTA amino acid sequences, which generally contain the same sequence variations (see Rizo and Gierasch (1992) Annu. Rev. Biochem. 61: 387); For example, the peptide can be produced by adding an internal cysteine residue capable of forming a disulfide bond between the molecules that is circularized. The amino acid sequence of the PD-L3 or VISTA polypeptide identified herein may allow a person skilled in the art to produce a polypeptide consistent with the PD-L3 or VISTA peptide sequence and its sequence variation. The polypeptide may be produced in prokaryotic or eukaryotic host cells by expressing a polynucleotide encoding the PD-L3 or VISTA peptide sequence, often as part of a larger polypeptide. Alternatively, the peptide can be synthesized by a chemical method. In recombinant hosts, methods for expression of heterologous polypeptides, chemical synthesis of polypeptides and in vitro translation are well known in the art. Peptide extension to specific amino-terminal and / or carboxy-terminal modifications and / or core sequences can provide physical, chemical, biochemical and pharmaceutical advantages, including, for example: enhanced safety, increased And / or efficacy, resistance to serum protease, favorable pharmacokinetic properties, and others. Peptides can be used therapeutically, for example, to treat diseases by altering co-stimulation in a patient.

The isolated PD-L3 or VISTA polypeptide, or a portion or fragment thereof, can be used as an immunogen to produce an antibody that binds to PD-L3 or VISTA, using common techniques for producing polyclonal and monoclonal antibodies . A PD-L3 or VISTA full-length polypeptide may be used, or the present invention provides an antigenic peptide portion of PD-L3 or VISTA for use as an immunogen. In some embodiments, the antigenic peptide of PD-L3 or VISTA comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2, 4 or 5 and comprises an epitope of PD-L3 or VISTA, Antibodies produced against peptides form specific immune complexes with PD-L3 or VISTA polypeptides. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, most preferably at least 30 amino acid residues. Preferred epitopes encompassed by antigenic peptides are those regions of PD-L3 or VISTA, as well as sites with high antigenicity, located in the extracellular domain of the polypeptide, e. G., The hydrophilic region.

A PD-L3 or VISTA immunogen is generally used to immunize an appropriate individual (e.g., rabbit, goat, mouse, or other mammal) using the immunogen. For example, suitable immunogenicity preparations may include recombinantly expressed PD-L3 or VISTA polypeptides or chemically synthesized PD-L3 or VISTA polypeptides. In addition, the preparation may comprise an adjuvant, for example a complete or incomplete Freund ' s adjuvant, or a similar immunostimulant. Immunization of appropriate individuals using immunogenic PD-L3 or VISTA preparation induces polyclonal anti-PD-L3 or VISTA antibody responses.

Thus, another aspect of the invention includes an anti-PD-L3 or VISTA antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule and a molecule comprising an antigen-binding site that specifically binds (immunoreacts) with an immunologically active site of an immunoglobulin molecule, such as PD-L3 or VISTA . Examples of immunologically active sites of immunoglobulin molecules include F (ab) and F (ab ') 2, which can be generated by treating an enzyme, such as pepsin, with an antibody. The present invention provides polyclonal and monoclonal antibodies that bind PD-L3 or VISTA molecules. The term "monoclonal antibody" or "monoclonal antibody composition ", as used herein, refers to an antibody comprising only one species of antigen binding site capable of immunoreacting with a particular epitope of PD-L3 or VISTA A group of molecules. Thus, monoclonal antibody compositions generally exhibit a single binding affinity for a particular PD-L3 or VISTA polypeptide that undergoes an immune response.

Polyclonal anti-PD-L3 or VISTA antibodies can be prepared by immunizing appropriate individuals with PD-L3 or VISTA immunogens, such as PD-L3 or VISTA-Ig fusion proteins, as described above. The anti-PD-L3 or VISTA antibody titer in the immunized individual can be measured by an enzyme linked immunosorbent assay (ELISA) using standard techniques, for example, fixed PD-L3 or VISTA Lt; / RTI > If desired, the antibody molecule for PD-L3 or VISTA can be isolated from mammals (e. G., From blood) and further purified by techniques well known in the art, for example protein A chromatography, a fraction can be obtained. At the appropriate time after immunization, for example, when the anti-PD-L3 or VISTA antibody titer is highest, the antibody-producing cells can be obtained from an individual and can be obtained using common techniques such as those described by Kohler and Milstein (1975) Nature 256 Yeh et al., (1976), J. Biol. Chem. 255: 4980-83, J. Immunol. 127: 539-46; Hybridoma technology described in Proc Natl. Acad. Sci. USA 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75, Cell hybridoma technology (Kozbor et al. (183) Immunol. Today 4:72), EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77-96) or by trioma techniques to produce monoclonal antibodies. Techniques for producing monoclonal antibody hybridomas are well known (see generally Kenneth, RH in Monoclonal Antibodies: A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York, NY (1980); Lemer, EA 1981) Yale J. Biol. Med. 54: 387-402; Gefter, ML et al. (1977) Somatic Cell Genet. 3: 231-36). Briefly, an immortal cell line (usually a myeloma) is fused with a lymphocyte (usually a splenocyte) from a mammal immunized with PD-L3 or VISTA immunogens, as described above, The resulting culture supernatant of the resulting hybridoma cells is screened to identify hybridomas producing monoclonal antibodies that bind to VISTA. Any of the many well-known protocols used to fuse fusion and immortalized cell lines can be applied for the purpose of producing anti-PD-L3 or VISTA monoclonal antibodies (Galfre, G. et al. (1977) Nature 266: 55052, see Gefter et al. (1977), Lerner (1981) and Kenneth (1980)). It will also be appreciated by those skilled in the art that there are many different ways that can be useful. Typically, the immortal cell line (e. G., A myeloma cell line) is derived from the same mammalian species as the lymphocyte. For example, mouse hybridomas can be generated by fusing lymphocytes from immortalized mouse new pimples and immunized mice using immunogenicity production of the present invention. A preferred immortal cell line is a mouse myeloma cell line sensitive to medium ("HAT medium") containing hypoxanthine, aminopterin and thymidine. Any of a number of myeloma cell lines such as P3-NSl / l-Ag4-l, P3-x63-Ag8.653 or Sp2 / 0-Agl4 myeloma can be used as a fusion partner according to the general techniques. The aquatic seed cell line is available from the ATCC. Generally, HAT-sensitive mouse myeloma cells are fused with mouse spleen cells using polyethylene glycol ("PEG"). The hybridoma cells made from the fusion are then sorted, which kill unfused and unproductively fused myeloma cells. (Unfused spleen cells die after several days because they have not been transformed). Hybridoma cells producing the single-claw antibodies of the present invention are detected by screening the hybridoma culture supernatant for antibodies that bind to PD-L3 or VISTA, for example, by using conventional ELISA assays.

Specific methods for producing antibodies that bind to PD-L3 or VISTA can be performed using methods described in the Examples and methods known in the art. An alternative method of producing a monoclonal antibody-secreted hybridoma is to use a recombinant combinatorial immunoglobulin library (e. G., An antibody phage display library) with a monoclonal anti-PD- Or VISTA, thereby isolating immunoglobulin library members that bind PD-L3 or VISTA. Kits for forming and screening phage display libraries are commercially available.

PD-L3 or VISTA protein, and / or affinity and / or affinity for PD-L3 or VISTA protein, and / or for binding to a specific epitope of PD-L3 or VISTA, such as an IgV domain or other specific domain. The antibody is rescreened to screen for antibodies. In addition, the antibody is screened in vitro and in vivo to identify modulation of specific functions and effects of PD-L3 or VISTA in immune and immune cells. For example, the test may be performed spontaneously by PD-L3 or VISTA, including cytokine production by CD4 + or CD8 + T cells, CD28 co-stimulation, CD4 + T cell proliferation and proliferation of naive and memory CD4 + PD-L3 < / RTI > or VISTA < / RTI > In a preferred embodiment, the test is carried out when the presence of PD-L3 or VISTA-Ig in vitro promotes inhibition by PD-L3 or VISTA-Ig, i.e. the immunosuppressive potential therapeutic anti- PD- May be performed to identify the antibody. The present invention relates to an anti-VSTA antibody binding to 136 amino acid extracellular domains, for example, 1-50, 50-100, 100-136 amino acids, an antibody specifically binding to IgV of VISTA, a stalk region, an antibody that specifically binds to the transmembrane region of VISTA, and an antibody that specifically binds to the cytoplasmic region of VISTA, and uses thereof. The specific site was identified in the present application.

Also included within the scope of the invention are recombinant anti-PD-L3 or VISTA antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human sites that can be made using common DNA techniques. The chimeric and humanized monoclonal antibodies may be produced by recombinant DNA techniques known in the art, for example, as described in Robinson et al., International Application No. < RTI ID = 0.0 > PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173, 494; Neuberger et al., PCT international application no. WO 86/01533; Cabilly et al., U.S. Pat. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (19-87) Cancer Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4: 214; Winter, U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyen et al. (1988) Science 239: 1534; And Beidler et al. (1988) J. Immunol. 141: 4053-4060.

An anti-PD-L3 or VISTA antibody (e. G., A monoclonal antibody) can be used to isolate PD-L3 or VISTA by common techniques, such as affinity chromatography or immunoprecipitation. PD-L3 or VISTA antibodies can facilitate the purification of native PD-L3 or VISTA from cells and the purification of recombinantly produced PD-L3 or VISTA expressed in host cells. In addition, anti-PD-L3 or VISTA antibodies can be used to detect PD-L3 or VISTA polypeptides (e. G., In cell lysates or cell supernatants) to investigate the abundance and expression pattern of PD-L3 or VISTA polypeptides. Lt; / RTI > Anti-PD-L3 or VISTA antibodies can be used to monitor polypeptide levels in tissues as part of a clinical test procedure, for example, to determine the effectiveness of a given treatment regimen. Detection may be facilitated by binding the antibody to a detectable substance. Examples of materials that can be detected include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; Examples of suitable complementary molecular monoclonal complexes include streptavidin biotin and avidin biotin; Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, Dansyl chloride or phycoerythrin; An example of a suitable luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin, and aequorin, and suitable radioactive materials include 1251, 1311, 35S or 3H.

III . Recombinant expression vectors and host cells

Another aspect of the invention includes vectors, preferably expression vectors, comprising nucleic acid molecules encoding PD-L3 or VISTA polypeptides (or portions thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other linked nucleic acid molecules. One type of vector is a "plasmid ", which refers to a circular double helix DNA loop in which additional DNA fragments can be ligated. Another form of vector is a viral vector, wherein the additional DNA portion can be linked to a viral genome. Certain vectors can be automatically replicated in host cells into which they are introduced (e. G., Bacterial vectors having a replication origin and an episomal mammalian vector). Other vectors (e. G., Non-episomal mammalian vectors) are introduced into the host cell and then integrated into the genome of the host cell, thus replicating along with the host genome. In addition, certain vectors may express operatively linked genes. This vector is referred to herein as an " expression vector. &Quot; In general, the utility of expression vectors in recombinant DNA techniques is often in the form of plasmids. In the specific description of the present invention, the "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used vector form. However, the present invention includes other forms of expression vectors, such as viral vectors (e. G., Defective retroviruses, adenoviruses and adeno-associated viral replication), which have the same function.

The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, wherein the recombinant expression vector operably linked to the nucleic acid sequence to be expressed is based on a host cell for expression Quot; means one or more control sequences selected. Within the recombinant expression vector, "operably linked" means that the target nucleotide sequence is capable of hybridizing to the nucleotide sequence of the host cell in a manner that enables expression of the nucleotide (e.g., in a transcriptional translation system in vitro or when the vector is introduced into the host cell , ≪ / RTI > "Regulatory sequences" are intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185: 3-7. Regulatory sequences include sequences of nucleotides that are constantly expressed in many kinds of host cells and nucleotide sequences that are expressed only in specific host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the transformed host cell, the level of expression of the desired protein and the like. Expression vectors of the invention can be introduced into host cells, thereby producing proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e. G., PD-L3 or VISTA polypeptides , PD-L3 or VISTA, fusion proteins or the like).

The recombinant expression vectors of the invention can be designed for the expression of PD-L3 or VISTA polypeptides in prokaryotic or eukaryotic cells. For example, PD-L3 or VISTA polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further described in Goeddel (1990). Alternatively, the recombinant expression vector may be transcribed or translated in vitro using, for example, the T7 promoter control sequence and T7 polymerase. For example, the purified fusion protein may be used in PD-L3 or VISTA activity assays (e.g., direct or competitive assays described below) or to produce antibodies specific for PD-L3 or VISTA polypeptides . In yet another embodiment, the PD-L3 or VISTA expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, PD-L3 or VISTA polypeptides can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for the expression of polypeptides cultured in insect cells (e.g., Sf9 cells) include pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and pVL series and Summers (1989) Virology 170: 31-39). In another embodiment, the nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When expressed in mammalian cells, the regulatory function of the expression vector is often provided by a viral regulatory element. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells see Sambrook, J. et al., Molecular Cloning chapters 16 and 17: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of favorably expressing the nucleic acid in a particular cell type (e.g., a tissue-specific modulator is used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific specific promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1988) Neuron-specific promoters (e. G., Neurofilament promoters; Byrne and < RTI ID = 0.0 > Pancreas-specific promoters (Edlund et al. (1985) Science 230: 912-916), and mammary-specific promoters (Ruddle (1989) Proc Natl. Acad. Sci. USA 86: 5473-5477) Mammal gland-specific promoters (e.g., milk whey promoter; US Pat. No. 4,873,316 and European Application No. 264,166). For example, developmentally-regulated promoters are also included, examples of which include murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and .alpha.-petato A protein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

The present invention also provides a recombinant expression vector comprising the DNA molecule of the present invention cloned as a recombinant vector in the antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in such a manner as to enable the expression (by transcription of a DNA molecule) of a DNA molecule that is antisense with PD-L3 or VISTA DNA. Regulatory sequences operably linked to nucleic acid molecules cloned in antisense orientation can be selected to continuously express antisense RNA molecules in various cell types. For example, the viral promoter and / or enhancer, or regulatory sequence, may be selected to constantly express the expression of the antisense RNA, or to regulate tissue specific or cell type specificity. The antisense expression vector may be in the form of a recombinant plasmid, a phagemid, or an attenuated virus in which the antisense nucleic acid is produced under the high-efficiency regulatory region and activity determined by the type of cell into which the vector is introduced . For a review of the regulation of gene expression using antisense genes, see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1 (1) 1986, incorporated herein by reference.

Another aspect of the present invention is the use of a PD-L3 or VISTA nucleic acid molecule of the invention, such as a PD-L3 or VISTA nucleic acid molecule in a recombinant expression vector, or a sequence that permits homologous recombination of the host cell's genomic site Lt; RTI ID = 0.0 > PD-L3 < / RTI > or VISTA nucleic acid molecule. The terms "host cell" and "recombinant host cell" are used interchangeably herein. The term encompasses not only certain individual cells but also progeny or potential progeny of such cells. Because the offspring may not be identical to the parent cell, but is included within the scope of the term used herein, as a particular variation may occur in successive generations due to environmental changes or environmental effects. The host cell may be prokaryotic or eukaryotic. Vector DNA can be introduced into prokaryotic or progressive cells via conventional transformation or transfection techniques. &Quot; Transformation "and " transduction ", as used herein, refers to various techniques known in the art for introducing an external nucleic acid (e. G., DNA) into a host cell, including calcium phosphate or calcium chloride co- phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection or electroporation. Suitable methods for the transfection or transformation of host cells are described in Sambrook et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other experimental manuals. In order to identify and screen these components, a selection marker (e.g., antibiotic resistance) is generally introduced into the host cell along with the target gene. Preferred screening markers include providing resistance to drugs such as G418, hygromycin and methotrexate. Prokaryotic or eukaryotic host cells in the host cells of the invention, e. G., A medium, can be used to produce (i. E. Express) PD-L3 or VISTA polypeptides. Accordingly, the present invention further provides methods for producing PD-L3 or VISTA polypeptides by using the host cells of the present invention. In certain embodiments, the method comprises culturing a host cell of the invention (host cell into which a recombinant expression vector encoding a PD-L3 or VISTA polypeptide is introduced) in a suitable medium, wherein the PD-L3 or VISTA polypeptide is produced . In yet another embodiment, the method further comprises isolating the PD-L3 or VISTA polypeptide from the medium or host cell.

In addition, the host cells of the invention can be used to produce non-human transgenic animals. For example, in certain embodiments, the host cells of the invention are modified into oocytes or embryonic stem cells into which PD-L3 or VISTA polypeptide-encoding sequences have been introduced. The host cells can then be used to produce non-transgenic transgenic animals or transgenic recombinant animals that have modified endogenous PD-L3 or VISTA sequences with exogenous PD-L3 or VISTA sequences introduced into their genome. The animal is useful for studying the activity and / or function of PD-L3 or VISTA, and for identifying and / or investigating modulation of PD-L3 or VISTA activity. As used herein, a "transduced animal" means a mammal such as a rodent such as a non-human animal, preferably a mammal, more preferably a rat or a mouse, wherein one or more of the animal cells contains a transgene lt; / RTI > Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. The transgene is an exogenous DNA integrated into the genome of the cell that remains in the genome of the animal in which the transgenic animal is developed and mature, thereby expressing the encoded gene product in one or more cell types or tissues of the transgenic animal do. As used herein, "homologous recombinant animal" means that an endogenous PD-L3 or VISTA gene is introduced into the endogenous gene and the animal's cells, for example, before the development of the animal, Preferably a mammal, more preferably a mouse, modified by homologous recombination between exogenous DNA molecules. The transgenic animal of the present invention can be obtained by introducing a PD-L3 or VISTA-encoding nucleic acid into a male pronuclei of a fertilized oocyte, for example by microinjection, retrovirus infection, To develop in a pseudopregnant female foster animal. The PD-L3 or VISTA cDNA sequence of SEQ ID NO: 1 or 4 can be introduced as an introduced gene into the genome of a non-human animal. Alternatively, a non-human homologue of the human PD-L3 or VISTA gene, such as a monkey or rat PD-L3 or VISTA gene, can be used as the transgene. Alternatively, a PD-L3 or VISTA gene homologue, such as another PD-L3 or VISTA family member, can be isolated based on hybridization to a PD-L3 or VISTA cDNA sequence of SEQ ID NO: 1 or 3 Desc / Clms Page number 2 > sub-part I) and can be used as transgene. Intron sequences and polyadenylation signals may also be included to increase the expression effect of the transgene. Tissue-specific regulatory sequences may be operably linked to the PD-L3 or VISTA transgene to cause expression of the PD-L3 or VISTA polypeptide in a particular cell. Methods for producing transgenic animals, particularly animals such as rats, through embryonic manipulation and microinjection are common in the art and are described, for example, in U.S. Pat. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al U.S. Pat. No. 4,873, 191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used to produce other transgenic animals. A transgenic founder animal can be identified based on the expression of PD-L3 or VISTA mRNA in the cells or tissues of the animal, or the presence of the PD-L3 or VISTA transgene in its genome. The transgenic animal may then be used to breed additional animals with the transgene. In addition, transgenic animals with transgenes encoding PD-L3 or VISTA polypeptides may be further propagated to other transgenic animals with other transgenes.

In order to produce a homologous recombinant animal, a vector containing a minimal portion of the PD-L3 or VISTA gene into which deletion, addition or substitution is introduced is produced, thereby transforming PD-L3 or VISTA, for example, functionally . The PD-L3 or VISTA gene may be a human or mouse gene (e.g., the cDNA of SEQ ID NO: 1 or 3).

In another embodiment, transgenic non-human animals can be produced that comprise a selected system that allows to regulate expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage PI (PI). For a description of the cre / loxP recombinase system, see Lakso et al. (1992) Proc Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinant enzyme system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 251: 1351-1355). If the cre / loxP recombinase system was used to regulate the expression of the transgene, an inducible gene encoding both the Cre recombinase and the selected polypeptide is essential. The animal may be produced through the production of a "double" transgenic animal, for example, two transgenic animals, one containing an introduced gene encoding the selected polypeptide, and the other animal containing the introduced gene encoding the recombinase .

Clones of non-human transgenic animals described herein are also described in Wilmut, I. et al. (1997) Nature 385: 810-813 and PCT international application Nos. ≪ / RTI > WO 97/07668 and WO 97/07669. Briefly, somatic cells from cells, for example transgenic animals, can be isolated and induced to enter the G0 phase out of the growth cycle. The quiescent cell can then be fused with an oocyte of the same species of animal from which the dormant cells have been isolated, for example through the use of electrical pulses. The reconstructed oocytes are then cultured, developed into morula or blastocyte stages, and then transplanted into fertile surrogate mother animals. The progeny of the surrogate animal may be a clone of the cell, e. G., An animal from which the somatic cell has been isolated.

IV . Pharmaceutical composition

PD-L3 or VISTA molecules (also referred to herein as "active compounds" or "modulating agents "), fragments of PD-L3 or VISTA molecules, such as PD-L3 or VISTA nucleic acid molecules, PD- Quot; may be included in a suitable pharmaceutical composition for administration. Such compositions typically comprise the nucleic acid molecule, polypeptide or antibody and a carrier such as a pharmaceutically acceptable carrier. As used herein, the term " Pharmaceutically acceptable carrier "as used herein refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and antifungal agents capable of pharmacological administration, Absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active materials is well known in the art. Any universal media or agent The Except that can not coexist with the active substance, it can be considered the use of it in the composition. There is also be included in the composition adjuvant active composition.

The composition may be administered in combination with a desired antigen, e. G., Tumor antigens, such as Toll like receptor agonists, type 1 interferons such as alpha and beta interferons, and the like, such as agonist CD40 antibodies and antibody fragments. CD40 agonists, preferably anti-human CD40 agonist antibodies and antibody fragments or other immunostimulatory agents or inhibitors such as PD-L1, PD-L2, CTLA4 fusion protein light specific antibodies .

In certain preferred embodiments, the composition or PD-L3 or VISTA-based treatment may further comprise an antigen or other immunologic agent. When an antigen is present in the composition or treatment, the antigen may be administered in combination with other components of the combination in an amount effective to form an immune response to the antigen. For example, the antigen may be administered in an amount of about 100 μg / kg to about 100 mg / kg. In certain embodiments, the antigen can be administered in an amount of about 10 μg / kg to about 10 mg / kg. In certain embodiments, the antigen can be administered in an amount of about 1 mg / kg to about 5 mg / kg. However, the specific amount of the antigen that is constituted in an amount effective to form an immune response may depend on a certain factor, such as the particular antigen administered; The particular agent to be administered and its amount; The state of the immune system; A method and order of administration of the agent and the antigen; The species to which the formulation is administered; And the desired treatment outcome. Thus, it is not practical to disclose the general amount, which consists of an effective amount of the antigen. However, those skilled in the art can easily determine an appropriate amount under consideration of the above factors.

The antigen may be any substance capable of generating a Th1 immune response, for example, a Th1 immune response may be a CD8 + T cell response, an NKT cell response, T cell response (.gamma ./. delta.T cell response), or one or more of the Thl antibody responses. Suitable antigens include, but are not limited to: Peptides; A polypeptide; Lipids; Glycolipids; Polysaccharides; Carbohydrates; Polynucleotides; Prions; Live or inactivated bacteria, viruses or fungi (live or inactivated bacteria, viruses or fungi); And bacterial, viral, fungal, protozoal, tumor-derived or organism-derived antigens, toxins or toxoids.

Also, certain currently available antigens, particularly recombinant proteins, glycoproteins, and peptides that do not generate a strong immune response, can be used in conjunction with the adjuvant combinations of the present invention. Exemplary experimental subunit antigens are those associated with viral diseases, such as adenovirus, AIDS, chicken pox, cytomegalovirus, dengue, feline leukemia feline leukemia, fowl plague, hepatitis A, hepatitis B, HSV-1, HSV-2, hog cholera, influenza A, influenza A, But are not limited to, influenza B, Japanese encephalitis, measles, parainfluenza, rabies, respiratory syncytial virus, rotavirus, wart, And yellow fever.

In certain embodiments, the antigen may be a cancer antigen or a tumor antigen. The terms cancerous and tumor antigens are used interchangeably and refer to antigens that are differentially expressed by cancer cells. Therefore, cancer antigens can be applied to differentially target immune responses to cancer cells. Thus, cancer antigens can potentially stimulate a tumor-specific immune response. Certain cancer antigens are not necessarily expressed, but are encoded by normal cells. Some of the antigens are characterized as being normally quiescent (i.e., not expressed) in normal cells, expressing at certain stages of differentiation, and transiently expressing (e. G., Embryonic and fetal antigens). Other cancer antigens include, for example, fusion proteins resulting from oncogenes (e.g., activated ras cancer genes), inhibitory genes (e.g., mutant p53), or internal deletions or chromosomal translocations Lt; RTI ID = 0.0 > mutant < / RTI > Other cancer genes can also be encoded by viral genes, such as those carried by RNA and DNA tumor viruses.

Examples of tumor genes include MAGE, MART-1 Melan-, gplOO, Dipeptidyl peptidase IV (DPPUV), adenosine deaminase-binding protein (ADAbp), cyclophilin b ), Colorectal associated antigen (CRC) -C017-1 A / GA733, carcinoembryonic antigen (CEA) and its antigenic epitope CAP-1 and CAP-2, etv6, am11, prostate (PSA) and its antigenic epitopes PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3- MAGE-A8, MAGE-A7, MAGE-A6, MAGE-A7, MAGE- MAGE-A9, MAGE-A10, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 -C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-system of tumor antigens (for example, GAGE-1 GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9), BAGE, RAGE, LAGE- 1, CDK4, tyrosinase, p53, MUC system, HER2 / neu, p21ras, RCAS 1, .alpha.-fetoprotein, .epsilon.- catenin, .beta.-catenin, .gamma.-catenin, pl20ctn, gplO.sup.pmel l17, .alpha.- catenin, , PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein; APC), fodrin, Connexin 37, Ig-idiotype, pi 5, gp75, GM2 and GD2 gangliosides, human papilloma viruses such as the proteins of virus proteins, Smad system of tumor antigens, Imp-1, PI A, EBV-encoded nuclear antigen (EBNA) -l, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and CT-7 and c-erbB-2.

Cancer or tumor and specific tumor antigens associated with (but not limited to) the above tumors include acute lymphoblastic leukemia (et a6, am l, cyclophilin b), B cell leukemia ), Glioma (E-cadherin, .alpha. -Catenin, .beta.-catenin, .gamma.-catenin, pl20ctn), bladder cancer (p21ras), biliary cancer ), Breast cancer (MUC family, HER2 neu, c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras, HER2 neu, c-erbB-2 , MUC family), colorectal cancer (Colorectal associated antigen (CRC) -C017-1A GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b) Gastric cancer (HER2 neu, c-erbB-2, ga733 glycoprotein), hepatocellular cancer (.alpha.-fetoprotein), Hodgkins lymphoma (Imp- l, EBNA-1) , Lung cancer (CEA, MAGE-3, NY-ESO-1)], lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan - A / MART-1, cdc27, MAGE-3, p21RAS, gpl00.sup.Pmel! 17], myeloma (MUC family, p21ras), non-small cell lung carcinoma (HER2 / neu, c-erbB-2), nasopharyngeal cancer (Imp- Prostate cancer (Prostate Specific Antigen (PSA) and its antigenic epitope PSA-1, PSA-1), ovarian cancer (MUC family, HER2 / neu, c-erbB, 2, and PSA-3, PSMA, HER2 / neu, c-erbB-2 and ga733 glycoprotein), renal cancer (HER2 neu, c-erbB-2), squamous cell carcinoma of the uterine cervix and esophagus testicular cancer (NY-ESO-1)], and T cell leukemia (T cell leukemia (HTLV-1 epitopes)), .

The pharmaceutical composition of the present invention is formulated so as to enable its administration route. Examples of routes of administration include, for example, parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal ) [Topical], transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: water for inhalation, saline solution, fixed oils, polyethylene glycol ( sterile diluents such as polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; Antibiotics such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; A buffer such as acetates, citrates or phosphates and an agent for tonicity control such as sodium chloride or dextrose. The pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water soluble) or suspensions, and sterile powders for the temporary preparation of sterile injectable solutions or suspensions. For intravenous administration, suitable carriers are physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that there is readily syringeability. It must be stable under the conditions of manufacture and storage and must be protected against contamination by the action of microorganisms such as bacteria and fungi. The carrier may be a solvent or a suspension medium, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like) and a suitable mixture thereof. The appropriate fluidity should be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of suspensions, and by the use of surfactants, May be made by various antibiotics and antibacterial agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be desirable to include isotonic agents in the composition, for example polyalcohol, such as sugar, mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition may be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

A sterile injectable solution may contain an active compound (e.g., a fragment of a PD-L3 or VISTA nucleic acid molecule, PD-L3 or VISTA polypeptide, an anti-PD-L3 or VISTA antibody, or an anti- PD- -PD-L1 antibody) together with one or a combination of the above-listed constituent substances in an appropriate solvent, and if necessary, filtering sterilization. Generally, suspensions are prepared by incorporating the active compound into a sterile vehicle, including a basic suspension medium and the other components required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of manufacture is vacuum drying and lyophilization, which provides the active ingredient with the desired additional components from its pre-sterilized filtrate solution to be.

In general, oral compositions generally include an inert diluent or a carrier for edible purposes. They can be enclosed in gelatin capsules or surrounded by tablets. For purposes of oral therapeutic administration, the active compound may be included with the additive and used in the form of a tablet, troche, or capsule. Oral compositions can also be prepared using a fluid carrier for use in mouth washing, wherein the composition in the fluid carrier is orally applied, spit or swallowed. Pharmaceutically compatible binders, and / or reinforcing materials may be included as part of the composition. Tablets, pills, capsules, trouche, and the like may include any of the following, or similar, compositions: binders such as microcrystalline cellulose, Gum tragacanth and gelatin; A disintegrating agent such as an excipient such as starch or lactose, alginic acid, Primogel, or corn starch; Lubricants such as magnesium stearate or Sterotes; Glidants such as colloidal silicon dioxide; A sweetening agent such as sucrose or saccharin; Or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compound may be formulated in the form of an aerosol spray or nebulizer from a suitable container or dispenser containing a suitable propellant, for example a gas such as carbon dioxide, .

In addition, systemic administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, a suitable penetrant to the penetrating barrier can be used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of a nasal spray. For transmucosal administration, the active compound may be formulated into ointments, salves, gels, or creams as is generally known in the art.

The compounds may also be formulated in the form of suppositories (for example, in conjunction with a universal suppository base such as cocoa butter and other glycerides) or retention enemas for rectal delivery have. In certain embodiments, the active compound is a controlled release formulation comprising a carrier that protects the compound against rapid removal from the body, for example, an implant and microencapsulated delivery systems, . Biodegradable and biocompatible polymers may be used. Examples of such polymers include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, , Polyorthoesters, and polylactic acid. Methods of preparing such formulations will be apparent to those skilled in the art. In addition, such materials (including liposomes targeting cells that have been fumed with monoclonal antibodies to viral antigens) can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, InC Liposomal suspensions, and can be used as a pharmaceutically usable carrier have. This is described, for example, by U.S. Pat. Pat. No. 4,522,811, which is known to those skilled in the art.

Formulation of dosage units into oral or parenteral compositions is particularly beneficial in terms of ease of administration and consistency of dosage. The dosage unit form used herein refers to an appropriate physical separation unit as an unified dose for the individual to be treated; Each unit contains a predetermined quantity of active compound that is calculated to produce the desired therapeutic effect associated with the essential pharmaceutical carrier. Specific descriptions for the dosage unit form of the invention are set forth and are directly dependent on the specific therapeutic effect to be achieved and the unique characteristics of the active compound and the inherent limitations that constitute the active compound for treating an individual.

The toxic and therapeutic effects of the compounds can be determined by cell culture or general pharmaceutical procedures in laboratory animals. The data obtained from the cell culture studies and animal studies can be used to determine the range of doses for use in humans. The dose of the compound preferably lies within a range around the concentration comprising less ED50 or no toxicity. The dose may vary within a range depending on the dosage form applied, and the administration route may be used. For the compounds used in the methods of the invention, the therapeutically effective dose can be measured early on from cell culture assays. The dose can be formulated in an animal model that is around a plasma concentration range that includes an IC50 determined in cell culture (i. E., The concentration of the test compound that achieves half the maximal inhibition of symptoms). The information can be used to determine more accurately the dose that is useful in humans. For example, levels in plasma can be measured by high performance liquid chromatography.

As defined herein, a therapeutically effective amount (i.e., an effective dose) of the protein or polypeptide is in the range of about 0.001 to 30 mg / kg body weight, preferably in the range of about 0.01 to 25 mg / kg body weight, Kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 20 mg / kg body weight, and still more preferably about 1 to 10 mg / kg, 6 mg / kg body weight. Those skilled in the art will understand that certain factors may affect the dose required to treat an individual effectively, including, for example, the severity of the disease or disorder, previous treatment, general health and / or age of the subject, will be. In addition, treating a subject in a therapeutically effective amount of a protein, polypeptide, or antibody may comprise a single treatment, and may preferably include a series of treatments.

In a preferred embodiment, the individual is administered for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4 weeks, 5 weeks or 6 injections Protein or polypeptide in a range of about 0.1 to 20 mg / kg body weight per week. It will also be appreciated that the effective dose of the antibody, protein or polypeptide used for treatment may be increased or decreased depending on the course of the particular treatment. The change in dose can result from the results of the diagnostic tests described herein and can be made clear.

The present invention includes agents that modulate the expression or activity of PD-L3 or VISTA. For example, the preparation may be a small molecule. For example, the small molecule may be an organic or inorganic molecule having a molecular weight of about 10,000 grams or less per mole, such as a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, a nucleotide, a nucleotide analog, Organic or inorganic compounds having a molecular weight less than or equal to about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than or equal to about 1,000 grams per mole, , Organic or inorganic compounds having a molecular weight of less than or equal to about 500 grams per mole, and salts, esters, and other pharmaceutically usable forms of such compounds. It is understood that the appropriate dose of small molecule formulation will depend on many factors involved in the knowledge of the healthcare practitioner, veterinarian or researcher of the art. For example, the capacity of the small molecule depends on the identity, size, and condition of the individual to be treated and the condition of the sample, and furthermore the route by which the composition is to be administered, It will vary depending on the effect the molecule desires to have.

Exemplary dosages include milligrams or micrograms of small molecular weight per kilogram of body or sample weight (e.g., from about 1 microgram per kilogram to about 500 per kilogram per kilogram) Milligram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is understood that the appropriate dose of a small molecule for the expression or activity to be modulated is determined by the efficacy of the small molecule. The appropriate capacity may be determined using the test described herein. When one or more of the small molecules is administered to an animal (e.g., a human) to modulate the expression or activity of a polypeptide or nucleic acid of the invention, for example, a healthcare professional, a veterinarian, A relatively low dose is prescribed, and then the dose is increased until appropriate response is seen. In addition, the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex and diet of the individual, the age and time of administration, the route of administration, the rate of excretion, The age of onset, the concurrent medication, and the degree of modulation or activity being regulated.

In addition, the antibody (or fragment thereof) may be conjugated with a therapeutic moiety, such as cytotoxicity, a pharmaceutically utilizable or radioactive metal ion. Cytotoxins or cytotoxic agents include any agent that is lethal to the cell. For example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide (e. G. etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione ), Mitoxantrone, mithramycin, actinomycin D, 1-dehydroestosterone, glucocorticoids, procaine, tetracaine tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic formulations include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, such as melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, Mitomycin C and cis-dichlorodiamine platinum (II) (cisplatin)), anthracyclines (for example, daunorubicin (see, for example, (Formerly daunomycin) and doxorubicin], antibiotics antibiotics [e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)], and anti- But are not limited to, anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugate of the present invention can be used to control a given biological response, and the drug moiety is not limited to conventional chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. For example, the polypeptide may comprise abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; The protein may be selected from the group consisting of tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, A tissue plasminogen activator; Biological response modulators include, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin- , "IL-6 ", granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) growth factors. These therapeutic moiety-binding techniques for antibodies are well known.

The nucleic acid molecule of the present invention can be inserted as a vector and used as a therapeutic vector gene. Gene therapy vectors may be used, for example, for intravenous infusion, local administration (see US Pat. No. 5,328,470) or stereotactic injection (see, for example, Chen et al. (1994) Proc Natl. Acad Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector may comprise a gene therapy vector in an available diluent or it may comprise a slow release matrix in which the gene delivery means is inserted. Alternatively, when the complete gene delivery vector, such as a retrovirus, is completely produced from the recombinant cell, the pharmaceutical preparation may include one or more cells that produce the gene delivery system. The pharmaceutical composition may be included in a container, a pack, or a dispenser together with the dosage regimen.

V. USES AND METHODS OF THE INVENTION

PD-L3 or VISTA molecules, such as the PD-L3 or VISTA nucleic acid molecules, polypeptides, polypeptide homologues, and antibodies and antibody fragments described herein, can be used in one or more of the following methods: a) screening assays; b) predictive medicine (eg, diagnostic tests, prognostic assays, monitoring clinical trials); And c) therapeutic methods (e.g., therapeutic and prophylactic to upregulate or downregulate the immune response). PD-L3 or VISTA polypeptides of the invention described herein have one or more of the following activities: 1) binds to and / or modulates its natural binding partner, 2) intercellularly or intracellularly Regulate signaling, 3) regulate the activation of T lymphocytes, and 4) regulate the immune response of organisms, such as mammals, such as mice or humans. For example, the isolated nucleic acid molecule of the invention can be used to express a PD-L3 or VISTA polypeptide (e. G., Through recombinant expression vectors in a host cell in gene therapy applications) and PD-L3 or VISTA mRNA Within the biological sample) or within the PD-L3 or VISTA gene, and can be used to modulate PD-L3 or VISTA activity, as described further below. The PD-L3 or VISTA polypeptide may be characterized by the production of a PD-L3 or VISTA inhibitor or an insufficient or excessive production of a PD-L3 or VISTA polypeptide. The PD-L3 or VISTA polypeptides also screen for naturally-occurring PD-L3 or VISTA binding partners and screen for drugs or compounds that modulate PD-L3 or VISTA activity and also include insufficient or excessive PD-L3 Or a disease or disorder characterized by the production of PD-L3 or VISTA having reduced, abnormal or unwanted activity as compared to the PD-L3 or VISTA wild-type polypeptide, such as the production of VISTA polypeptides Systemic disorders include severe combined immunodeficiency, multiple sclerosis, systemic lupus erythematosus, type I diabetes mellitus, lymphoproliferative syndrome, inflammatory bowel disease, Inflammatory bowel disease, allergies, asthma, graft-versus-host disease, And transplant rejection; Immune responses to infectious pathogens such as bacteria and viruses; And immune system cancers such as lymphoma and leukemia. In addition, anti-PD-L3 or VISTA of the present invention can detect or isolate PD-L3 or VISTA polypeptides, modulate the biological activity of PD-L3 or VISTA polypeptides, and modulate PD-L3 or VISTA activity, For example by modulating the mutual coupling between PD-L3 or VISTA and its natural binding partner.

A. Screening test:

The present invention relates to the use of PD-L3 or VISTA and its natural or synthetic inhibitors, such as PD-L3 or VISTA expression or PD-L3 or VISTA activity, (Also referred to herein as a " screening test ") for identifying candidate or test compounds or agents (peptides, peptidomimetics, small molecules or other drugs) that have a stimulatory or inhibitory effect on the interaction between binding partners, .

In certain embodiments, the invention relates to the ability of a candidate or a test compound to bind to a PD-L3 or VISTA protein or polypeptide or a biologically active site thereof, such as a PD-L3 or VISTA polypeptide, Lt; RTI ID = 0.0 > and / or < / RTI > In yet another embodiment, the invention provides a method for screening a candidate agent or a test compound that binds to or modulates the activity of a PD-L3 or VISTA protein or polypeptide or biologically active site thereof. In a preferred embodiment, the invention relates to a method of stimulating (or stimulating) immune function in immune function that is phonetically regulated by PD-L3 or VISTA based on the effect on the mutual binding between PD-L3 or VISTA and its natural binding partner Or a test for screening candidates or test compounds with inhibitory effects. The PD-L3 or VISTA related functions inhibit the production of cytokines (e.g., U-2, T-cell gamma interferon), inhibit moderate CD28 co-stimulation, inhibit CD4 + and CD8 + T cell proliferation, Inhibition of proliferation of naive and memory CD4 + T cells, and inhibition of TCR activation without inducing apoptosis. The test compounds of the present invention can be obtained by using any of the many approaches in combinatorial library methods known in the art, including biological libraries; Spatially addressable parallel solid phase or solution phase libraries; Synthetic library methods that require deconvolution; One-bead one-compound library method; And synthetic library methods using affinity chromatography selections. While the biological library approach is limited to peptide libraries, the other four approaches are applicable to peptides, non-peptide oligomers or small molecules of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12: 145).

In certain embodiments, the assay comprises the steps of contacting a cell expressing a PD-L3 or VISTA polypeptide or a biologically active site thereof with a test compound and determining the ability of the test compound to modulate PD-L3 or VISTA activity Based cell-based assay. Determining the ability of the test compound to modulate PD-L3 or VISTA activity can be accomplished, for example, by binding to its natural binding partners of PD-L3 or VISTA and monitoring its ability to modulate immune cell activity. The immune cells may be, for example, T cells, B cells or myeloid cells. Determining the ability of the test compound to modulate a counter-receptor (to be determined) and PD-L3 or VISTA binding can be accomplished, for example, by determining the ability of the T-cell expressing the PD-L3 or VISTA counter- And PD-L3 or VISTA with a radioisotope or enzymatic label to modulate the binding of PD-L3 or VISTA. The ability of the test compound to bind to PD-L3 or VISTA can be determined, for example, by conjugating the compound with a radioisotope or an enzymatic label, whereby the binding of PD-L3 or VISTA to the compound, Lt; RTI ID = 0.0 > PD-L3 < / RTI >

It is also within the scope of the present invention to determine the ability of a compound to cross-link with PD-L3 or VISTA without any labeling of the linker. For example, a microphysiometer can be used to detect compound interactions (McConnell, HM et al. (1992) Science 257: 1906-1912) without the labeling of the compound or microfluidometer. . As used herein, the term " microphysiometer "(e.g., a cytosensor) refers to the rate at which a cell oxidizes its environment using a light-addressable potentiometric sensor (LAPS) This change in acidification can be used as an indicator of the interactions between the compound and PD-L3 or VISTA.

In yet another embodiment, the assay comprises contacting a T cell expressing a PD-L3 or VISTA binding partner with a test compound and modulating (e. G., Stimulating or modulating the activity of the PD-L3 or VISTA binding partner of the test compound Inhibition) of the cell-based test. Determining the ability of the test compound to modulate the activity of the PD-L3 or VISTA binding partner, for example, the ability to bind PD-L3 or a VISTA polypeptide with a PD-L3 or VISTA binding partner, ≪ / RTI >

Determining the ability to bind or interact with a PD-L3 or VISTA polypeptide, or a biologically active fragment thereof, of a PD-L3 or VISTA binding partner is accomplished by one of the methods described above that determines direct binding . In a preferred embodiment, determining the ability to bind to or interact with a PD-L3 or VISTA binding partner of PD-L3 or VISTA can be accomplished by determining the activity of said binding partner. For example, the activity of the binding partner may be determined by detecting induction of a cellular second messenger (e.g. tyrosine kinase or phosphatase activity), detecting the catalytic / enzymatic activity of a suitable substrate , Detecting the induction of a receptor gene (including, for example, a target-response modifier operably linked to a nucleic acid encoding a detectable marker such as luciferase), or detecting a target-regulated cellular response have. For example, determining the ability to bind or interact with a native PD-L3 or VISTA binding partner of a PD-L3 or VISTA polypeptide can be accomplished by measuring inhibition in the ability of the compound to modulate immune cell co- Or by interfering with the ability of PD-L3 or VISTA to bind to antibodies that recognize PD-L3 or portions of VISTA polypeptides. In certain embodiments, a compound that modulates T cell activation can be identified by modulating the ability of the compound to modulate T cell proliferation or cytokine production. In a preferred embodiment, a compound that modulates T cell activation can be identified by determining the ability of the compound to modulate T cell proliferation or cytokine production at one or more antigen concentrations.

In another embodiment, the test of the present invention involves the ability of a PD-L3 or VISTA polypeptide or biologically active site thereof to contact a test compound and to bind a PD-L3 or VISTA polypeptide or a biologically active site thereof It is a cell-glass test, as determined. A preferred biologically active portion of a PD-L3 or VISTA polypeptide that can be used in the assays of the invention is a cell that binds to a fragment that participates in a mutual binding with a non-PD-L3 or VISTA molecule, for example, a PD-L3 or VISTA binding partner Gt; domain < / RTI > The binding of the PD-L3 or VISTA polypeptide to the test compound can be determined either directly or indirectly as described above.

In another embodiment, the assay comprises contacting a PD-L3 or VISTA polypeptide or a biologically active portion thereof with a test compound and modulating the activity of the PD-L3 or VISTA polypeptide or a biologically active site thereof Cell-free assay that determines the ability of the test compound to stimulate, inhibit, stimulate, or inhibit cell growth. Determining the ability of the test compound to modulate the activity of a PD-L3 or VISTA polypeptide can be accomplished, for example, by PD-L3 binding to PD-L3 or VISTA binding partner by one of the methods described above for determining direct binding Or by determining the ability of VISTA. The cell-free assay of the present invention can be used in both soluble and / or membrane-bound forms of polypeptides (e. G., PD-L3 or VISTA polypeptides or biologically active sites thereof, or binding partners that bind PD-L3 or VISTA) . In the case of a cell-free assay in which a membrane-bound polypeptide form is used (e.g., the cell membrane PD-L3 or VISTA), it is preferable to use a solubilizing agent so that the membrane-bound form of the polypeptide can be retained in the solution Do. Examples of the solubilizing agent include n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton RTM X-100, Triton RTM X-114, (ethylene glycol ether) n], 3 - [(3-chloramidopropyl) dimethylamino] -1-propanesulfonate (hereinafter referred to as " isotridecypoly (3- [(3-chloramidopropyl) dimethylaminio] -2-hydroxy-1-propanesulfonate (CHAPS) (3-cholamidopropyl) dimethylamminio] -2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl, dbd.N, N-dimethyl-3-amonio-1-propanesulfonate Nonionic detergents such as N, N-dimethyl-3-ammonio-1-propane sulfonate (no n-ionic detergent.

In one or more embodiments of the test method of the present invention, PD-L3 or VISTA or < RTI ID = 0.0 > PD-L3 < / RTI > or < RTI ID = 0.0 > It may be desirable to immobilize its binding partner. The binding of the PD-L3 or VISTA to the auditing compound, or the interaction of the PD-L3 or VISTA polypeptide with its binding partner in the presence and absence of the candidate compound, may be in a vessel suitable for containing said agent. Examples of such vessels include microtitre plates, test rubes, and micro-centrifuge tubes. In certain embodiments, the fusion protein may be provided with the addition of a domain that allows one or both of the polypeptides to be bound to the matrix. For example, a glutathione-S-transferase PD-L3 or VISTA fusion protein or a glutathione-S-transferase binding partner fusion protein may be conjugated to glutathione sepharose beads or glutathione (Sigma Chemical, St. Louis, Mo), and then the test compound or the test compound and the non-absorbable binding partner polypeptide or PD-L3 or VISTA polypeptide , And the mixture is cultured under conditions that induce complex formation (e. G., PH and physiological conditions for salts). Following incubation, the beads or microtitre plate wells are washed to remove unbound elements, the matrix is fixed in the case of beads, complex formation is carried out directly or indirectly, for example, as described above We decide together. Alternatively, the complex can be separated from the matrix, and the level or activity of PD-L3 or VISTA binding can be determined using conventional techniques. Other techniques for immobilizing polypeptides in the matrix can be used in the screening test of the present invention. In yet another embodiment, determining the ability of the test compound to modulate the activity of a PD-L3 or VISTA polypeptide comprises determining the ability of the test compound to modulate the activity of a molecule that functions downstream of PD-L3 or VISTA For example, by interacting with the cytoplasmic domain of the PD-L3 or VISTA binding partner. For example, the level of the secondary messenger, the activity of the interacting molecule in the appropriate target, or the binding of the mutual binding partner to a suitable target can be determined as described above.

In another embodiment, the modulator of PD-L3 or VISTA expression can be identified in a manner wherein the cell is contacted with a candidate compound and the expression of PD-L3 or VISTA mRNA or polypeptide in the cell is determined. The level of expression of PD-L3 or VISTA mRNA or polypeptide in the presence of the candidate compound is compared to the level of PD-L3 or VISTA mRNA or polypeptide expression in the absence of the candidate compound. The candidate compound can then be identified as a modulator of PD-L3 or VISTA expression based on this comparison when the change is statistically significant.

In yet another embodiment, the PD-L3 or VISTA polypeptide comprises another polypeptide ("PD-L3 or VISTA-binding protein", "PD-L3 or VISTA binding partner") that binds or interacts with PD- Quot; bait protein " in a two-hybrid assay or in a 3-hybrid assay to identify other polypeptides related to the activity of PD-L3 or VISTA-bp & (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; The PD-L3 or VISTA-binding protein can also be produced by the method described in Bott et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696 and Brent WO94 / As a downstream element of a PD-L3 or VISTA-mediated signal transduction pathway, a signal of a PD-L3 or VISTA polypeptide or of a signal by a PD-L3 or VISTA target The PD-L3 or VISTA-binding polypeptide may be a PD-L3 or VISTA inhibitor. The Te 2 -hybrid system is based on the regulatory characteristics of most transcription factors, In one construct, the PD-L3 or VISTA polypeptide is encoded by a gene that encodes a known transcription factor, such as a transcription factor, In another embodiment, an unidentified polypeptide ("prey" or "sample") from a library of DNA sequences is linked to a gene encoding the DNA binding domain of a factor (eg, GAL-4) ) "] DNA sequence encoding the DNA sequence is linked to a gene encoding the activation domain of the known transcription factor. If the "bait" and the "prey" polypeptide can interact to form a PD-L3 or VISTA-dependent complex in vivo, the DNA-binding and activation domains of the transcription factor will be in close proximity. This proximity allows transcription of the operably linked receptor gene (e. G., LacZ) to transcriptional factors to respond to transcription factors. Expression of the receptor gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain a cloned gene encoding the polypeptide interacting with PD-L3 or VISTA polypeptides have.

In another aspect, the invention includes two or more combinations of the tests described herein. For example, modulators can be identified using cell-based or cell-free assays, and the ability to modulate the activity of PD-L3 or VISTA polypeptides of the agent can be determined in vivo, for example, / RTI > and / or animal models for tumorigenesis. ≪ RTI ID = 0.0 >

Further, the present invention includes novel agents identified by the screening tests described above. Thus, the use of an agent identified in the appropriate animal model described herein is within the scope of the present invention. For example, the identified agent described herein (e.g., PD-L3 or VISTA modulator, antisense PD-L3 or VISTA nucleic acid molecule, PD-L3 or VISTA-specific antibody, or PD-L3 or VISTA binding partner ) Can be used in animal models to determine the effect, toxicity or side effect of treatment with the agent. Alternatively, the agent identified as described herein can be used in animal models to determine the mechanism of action of the agent. The invention also encompasses the use of a new agent identified by the screening test described above for treatment, as described herein.

B. Detection Assay

Sites or fragments of the cDNA sequences identified herein (and corresponding corresponding complete gene sequences) can be used in a variety of ways as polynucleotide reagents. For example, the sequences may be used to: (i) map each of their genes to a chromosome and thus localize the gene site associated with the genetic disease; (ii) identify individuals from trace amounts of biological samples (tissue typing); And (iii) forensic identification of biological samples. This application is described in the following subsections.

1. Chromosome Mapping

Once the sequence (or portion of the sequence) of the gene is isolated, this sequence can be used to map the position of the gene in the chromosome. This process is called chromosomal mapping. Thus, a site or fragment of the PD-L3 or VISTA nucleotide sequence described herein can be used to map the location of the PD-L3 or VISTA gene on the chromosome. Mapping of the PD-L3 or VISTA sequence to the chromosome is an important first step in that the sequence is correlated with genes associated with the disease. Briefly, the PD-L3 or VISTA gene can be mapped to the chromosome by producing a PCR primer (preferably 15-25 bp in length) from the PD-L3 or VISTA nucleotide sequence. Computer analysis of PD-L3 or VISTA sequences can be used to predict primers that do not lie in one or more exons in genomic DNA, thus complicating the amplification process. The primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. PD-L3 < / RTI > or VISTA sequence. Somatic hybrids can be produced by fusing somatic cells (e.g., human and mouse cells) from different mammals. As hybrids of human and mouse cells grow and divide, they gradually lose the human chromosome in a random order, but retain the mouse chromosome. By using a medium in which mouse cells can not grow because there is no specific enzyme and only human cells can grow, the human cell chromosome containing the gene encoding the necessary enzyme will be retained. By using various media, a panel of hybrid cell lines can be established. Each cell line in the panel allows easy mapping of each gene into a specific human chromosome, including a single human chromosome or a small number of human chromosomes, and a whole set of mouse chromosomes (D'Eustachio, P. et al (1983) Science 220: 919-924). In addition, somatic cell hybrids containing only fragments of human chromosomes can be produced by using human chromosomes with translovation and deletion.

PCR mapping of somatic cell hybrids is a fast process for assigning specific sequences to specific chromosomes. Three or more sequences can be run for a day using a single thermal cycler. By using PD-L3 or VISTA nucleotide sequences designed for oligonucleotide primers, sublocalization can be accomplished using panels of fragments from specific chromosomes. Other mapping strategies that can be used to similarly map PD-L3 or VISTA sequences to their chromosomes include in situ hybridization (Fan, Y. et al. (1990) Proc Natl. Acad Sci. USA 87: 6223-27), pre-screening using labeled flow-sorted chromosomes, and pre-screening using labeled flow-sorted chromosomes and pre-screening by hybridization to chromosome- pre-selection.

It can be used to provide an accurate location of chromosomes in one step of fluorescence in situ hybridization (FISH), a fluorescence of the DNA sequence for mid-term chromosome dispersion. Chromosome spread can be made using cells that have been stalled in the metaphases by chemicals such as colcemids that destroy the mitotic spindle. The chromosome is briefly treated with trypsin and then stained with Giemsa. The appearance of bright and dark bands in each chromosome is developed, and the chromosomes can be individually identified. The FISH technique can be used with DNA sequences as short as 500 or 600 bases. However, clones over 1,000 bases have a high probability of combining with a particular chromosomal location, indicating sufficient signal strength for simple detection. Preferably 1,000 bases, more preferably 2,000 bases, are sufficient to obtain good results over a reasonable time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988). Reagents for chromosomal mapping can be used individually to display a single chromosome or a single site on the chromosome, or a panel of reagents can be used to display multiple sites and / or multiple chromosomes. Reagents corresponding to virtually unencrypted sites of the gene are preferred in the mapping process. Encoding sequences are more conserved in the gene system, thus increasing the chance of cross hybridization during chromosome mapping.

Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome may be related to genetic map data. Ultimately, complete sequence analysis of genes from several individuals can be performed to confirm the presence of the mutation and to distinguish mutations from the polymorphosm.

2. Tissue typing

In addition, the PD-L3 or VISTA sequences of the invention can be used to identify individuals from trace amounts of biological samples. In addition, the sequences of the present invention can be used to provide a representative technique for determining the actual base-sugar-base DNa sequence in a selected portion of an individual genome. Thus, the PD-L3 or VISTA nucleotide sequences described herein can be used to prepare two PCR primers from the 5 ' and 3 ' ends of the sequence. These primers can then be used to amplify individual DNA and then reveal their sequence.

A panel of individual DNA sequences prepared in this manner provides individual identification of individual individuals since each individual has a unique set of DNa sequences due to allelic phase heterogeneity. The sequences of the present invention can be used to obtain sequence identity from an individual or tissue. The PD-L3 or VISTA nucleotide sequences of the invention specifically represent portions of the human genome. Allelic variants occur somewhat at the coding site of the sequence and occur more at the unencrypted site. The variation of alleles among individual humans is estimated to occur at approximately one frequency per 500 bases. Each of the sequences described herein can be used to some extent as a companion to the ability to compare DNA from an individual for identification purposes. Because more polymorphisms occur at noncoding sites, fewer sequences are required to distinguish individuals. The unencrypted sequence of SEQ ID NO: 1 or 4 readily provides a positive individual identification using panels of approximately 10 to 1,000 primers, each of which provides an unencrypted sequence of 100 bases can do. If a coding sequence such as the sequence in SEQ ID NO: 3 or 6 is predicted, a more suitable number of primers for positive individual identification would be 500-2000.

When a panel of reagents from the PD-L3 or VISTA nucleotide sequences described herein is used to generate an identification database for an individual, the same reagent can be used subsequently to identify tissue from the individual have. Using a special identification database, living or dead, individual positive identities can be made from dramatically smaller tissue samples.

3. Use of PD-L3 or VISTA sequences in Forensic Biology

In addition, DNA-based identification techniques can be used in forensic biology. The sequences of the present invention may be used to provide polynucleotide sequences, such as PCR primers, that target specific loci in the human genome, which may include, for example, another "identification market" That is, it can increase the reliability of DNA-based forensic identification by providing another DNA sequence that is specific to a particular individual. As mentioned above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the aspect formed by the restriction enzyme generated fragment. Sequences targeting an unencrypted region of SEQ ID NO: 1 or 3 are particularly suitable for such uses because polymorphisms occur more frequently in unencrypted sites, making it easier to distinguish individuals using the techniques described above. Examples of polynucleotide reagents are those derived from a non-coding region of SEQ ID NO: 1 or 3 having a PD-L3 or VISTA nucleotide sequence or a portion thereof, for example at least 20, preferably at least 30 bases in length Lt; / RTI > The PD-L3 or VISTA nucleotide sequence described herein may further be used to provide a labeled or labeled probe that can be used in in situ hybridization techniques to identify specific tissues such as polynucleotide reagents, e.g., lymphocytes . This can be very useful when forensic pathologists represent an organization of unknown origin. The PD-L3 or VISTA probe panel may be useful for identifying tissue by species and / or by species of origin. In a similar manner, the reagents, such as PD-L3 or VISTA primers or probes, may be useful for screening tissue culture for contamination (i. E. Screening for the presence of a mixture of different species of cells in culture).

C. Predictive Medicine

The present invention also encompasses predictive medicine for prophylactic treatment of individuals by using diagnostic tests, prognostic assays, and clinical trials for purposes of prediction (prediction). Thus, one aspect of the present invention is to determine PD-L3 or VISTA activity as well as PD-L3 or VISTA polypeptide and / or nucleic acid molecules in a biological sample (e.g., blood, serum, Or a disease or disorder associated with the expression or activity of unwanted PD-L3 or VISTA, or a diagnostic test for determining whether the disease is at risk of developing. The invention also provides an anticipatory (or predictive) test to determine if a person is at risk of developing a PD-L3 or VISTA polypeptide, a disease associated with nucleic acid expression or activity. For example, mutations in PD-L3 or VISTA genes can be examined in biological samples. The test may be used for prophylactic or predictive purposes that may proactively treat an individual prior to the onset of a PD-L3 or VISTA polypeptide, a nucleic acid expression or activity associated with or characterized by the nucleic acid expression or activity.

Another aspect of the invention includes monitoring the effects of agents (e. G., Drugs, compounds) in the expression or activity of PD-L3 or VISTA in clinical trials. These and other formulations are described in more detail in the following sections.

1. Diagnostic tests

An exemplary method for detecting the presence or absence of a PD-L3 or VISTA polypeptide or nucleic acid comprises obtaining a biological sample from an inspecting subject and contacting the biological sample with a PD-L3 or VISTA polypeptide or a polypeptide of PD-L3 or VISTA (E. G., MRNA or genomic DNA) encoding the nucleic acid encoding the PD-L3 or VISTA polypeptide or nucleic acid encoding the PD-L3 or VISTA polypeptide or nucleic acid in the biological sample. A preferred agent for detecting PD-L3 or VISTA mRNA or genomic DNA is a labeled nucleic acid probe that can hybridize with PD-L3 or VISTA mRNA or genomic DNA. For example, the nucleic acid probe may be a PD-L3 or VISTA nucleic acid as disclosed in SEQ ID NO: 1 or 3, or a portion thereof, such as at least 15, 30, 50, 100, 250 or 500 nucleotides in length And may be an oligonucleotide sufficient to specifically hybridize with PD-L3 or VISTA mRNA or genomic DNA under certain conditions. Other suitable probes for use in the diagnostic tests of the present invention are described herein. A preferred agent for terminating a PD-L3 or VISTA polypeptide is an antibody, preferably a detectable label, capable of binding to a PD-L3 or VISTA polypeptide. The antibody may be a polyclonal, or more preferably a monoclonal. A whole antibody, or a fragment thereof (e.g., Fab or F (ab ') 2) can be used. The term "labeled " with respect to the probe or antibody encompasses not only the direct labeling of the probe or antibody, but also the direct labeling of another reagent, by binding the detectable substance to the probe or antibody (i.e., And indirect labeling of the probe or antibody by the reactivity used. Examples of indirect labeling include detection of DNA end-labeling with biotin detectable using a primary antibody using a fluorescently labeled secondary antibody and fluorescently labeled streptoavidin. The term "biological sample" includes tissues, cells and biological fluids isolated from an individual, as well as tissues, cells, and fluids present in the individual. That is, the detection method of the present invention can be used to detect PD-L3 or VISTA mRNA, polypeptide or genomic DNA in a biological sample as well as in vitro, in vivo. For example, in vitro techniques for detection of PD-L2 mRNA include Northern hybridizations and in situ hybridization. In vitro techniques for detection of PD-L3 or VISTA polypeptides include enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence . In vitro techniques for detection of PD-L3 or VISTA genomic DNA include Southern hybridization. In addition, in vivo techniques for detecting PD-L3 or VISTA polypeptides include introducing the labeled anti-PD-L3 or VISTA antibody as an entity. For example, the antibody can be labeled with a radiolabeled marker whose presence and location in the can be detected by conventional imaging techniques. In some embodiments, the biological sample comprises polypeptide molecules from the test subject. Alternatively, the biological sample may comprise the test organism or mRNA molecule, or a genomic DNA molecule from the test organism. A preferred biological sample is a serum sample separated by a universal means from an individual. In yet another embodiment, the method further comprises obtaining a control biological sample from a control individual, wherein the presence of the PD-L3 or VISTA polypeptide, PD-L3 or VISTA polypeptide, mRNA, or genomic DNA, Contacting a compound or agent capable of detecting mRNA or genomic DNA so that it can be detected in the sample and detecting the presence of PD-L3 or VISTA polypeptide, mRNA, or genomic DNA in the control sample in a PD -L3 or VISTA polypeptide, mRNA, or genomic DNA.

The invention also includes a kit for detecting the presence of PD-L3 or VISTA in a biological sample. For example, the kit may comprise a labeled compound or agent capable of detecting a PD-L3 or VISTA polypeptide or mRNA of a biological sample; Means for determining the amount of PD-L3 or VISTA in said sample; And means for comparing the amount and standard of PD-L3 or VISTA in the sample. The compounds and formulations may be packaged in a suitable container. The kit may also include instructions for using a kit to detect PD-L3 or VISTA polypeptides or nucleic acids.

2. Predictive inspection

May be used to identify individuals who have or are at risk of developing the diagnostic methods described herein, as well as diseases or disorders associated with abnormal or unwanted PD-L3 or VISTA expression or activity. As used herein, the term "abnormal" includes PD-L3 or VISTA expression or activity, deviating from wild-type PD-L3 or VISTA expression or activity. Abnormal expression or activity includes increased expression or activity, as well as expression or activity not following the expression pattern of wild-type development or the subcellular pattern of expression. For example, in the case of mutations in PD-L3 or VISTA causing abnormal PD-L3 or VISTA expression or activity to be PD-L3 or VISTA genes that are under-expressed or over-expressed and that the mutation is non-functional PD-L3 VISTA polypeptide or a polypeptide that does not function in a wild-type manner, such as a polypeptide that does not interact with a PD-L3 or VISTA binding partner, or a polypeptide that interacts with a non-PD-L3 or VISTA binding partner do. As used herein, the term "unwanted" includes unwanted phenomena associated with biological reactions such as immune cell activation. For example, the term unwanted includes PD-L3 or VISTA expression or activity which is undesirable in the individual.

The tests described herein, such as the diagnostic tests or the following tests, may be used for the treatment of diseases associated with erroneous modulation of PD-L3 or VISTA polypeptide activity or nucleic acid expression, such as autoimmune diseases, immune deficiency diseases, Can be used to identify individuals at risk for developing a disease, allergic or inflammatory bowel or cancer. Thus, the present invention relates to a method for detecting abnormal or undesired PD-L3 or VISTA expression or activity, wherein said test sample is obtained from an individual and wherein PD-L3 or VISTA polypeptide or nucleic acid (e.g., mRNA or genomic DNA) Wherein the presence of a PD-L3 or VISTA polypeptide or nucleic acid has the risk of developing a disease or disorder associated with abnormal or unwanted PD-L3 or VISTA expression or activity It is diagnostic for an individual. As used herein, "test sample" means a biological sample obtained from an individual of the target. For example, the test sample may be a biological fluid (e. G., Cerebrospinal fluid or serum), a cell sample, or a tissue.

In addition, the anticipatory test described herein may be used to treat a subject (e.g., an agent, an antagonist, a peptidomimetic, a peptidomimetic, an antagonist, an antagonist, A polypeptide, a peptide, a nucleic acid, a small molecule, or other drug candidate). For example, the methods can be used to determine whether an individual can be effectively treated for an autoimmune disorder, an immune deficiency disorder, an immune system cancer, or an allergic or inflammatory disorder. Accordingly, the present invention provides a method for determining whether an individual can be effectively treated with an agent for disorders associated with abnormal or unwanted PD-L3 or VISTA expression or activity, wherein the test sample is obtained and the PD-L3 or VISTA polypeptide or nucleic acid expression or activity is detected (e. G., Where the presence of PD-L3 or VISTA polypeptide or nucleic acid expression or activity is detected in an agent that treats disorders associated with abnormal or unwanted PD-L3 or VISTA expression or activity Lt; RTI ID = 0.0 > (e. G., ≪ / RTI > In addition, the methods of the present invention can be used to detect genetic alterations in the PD-L3 or VISTA gene, so that individuals with the altered gene are characterized by PD-L3 or VISTA polypeptide activity or erroneous regulation in nucleic acid expression For example, autoimmune disorders, immunodeficiency disorders, immune system cancers, allergic disorders, or inflammatory disorders. The method described herein can be performed, for example, by using a pre-packaged diagnostic kit comprising at least one probe nucleic acid or antibody reagent described herein, which can be used, for example, in PD-L3 Or to diagnose a patient having a history or family history of a disease or disease associated with the VISTA gene. In addition, any cell type or tissue in which PD-L3 or VISTA is expressed can be used in the predictive tests described herein.

3. Monitoring effects during clinical trials

Monitoring the effects (e. G., Modulation of cellular proliferation and / or migration) of agents (e. G., Drugs) in the expression or activity of PD-L3 or VISTA polypeptides can be applied not only to basic drug screening but also to clinical trials. For example, the efficacy of the agent, which increases PD-L3 or VISTA gene expression, polypeptide levels, or upregulates PD-L3 or VISTA activity, may result in reduced PD-L3 or VISTA gene expression, RTI ID = 0.0 > PD-L3 < / RTI > or VISTA activity. Or PD-L3 or VISTA gene expression, decrease the polypeptide level or down-regulate the PD-L3 or VISTA activity by increasing the PD-L3 or VISTA gene expression, the polypeptide level, Or in clinical trials of individuals exhibiting PD-L3 or VISTA activity. As described, PD-L3 or VISTA is expressed in many types of plastic cells, including APCs (macrophages and myeloid dendritic cells), and CD4 + T cells, and more specifically CD1lc + DCs, CD4 + T cells (Foxp3- (Including both functional T cells and Foxp3 + n T regs), CD8 + T cells, and Grl + granulocytes, and are expressed at low levels in B and NK cells. In this clinical trial, PD-L3 or VISTA-related disorders, such as the expression or activity of PD-L3 or VISTA genes, and more preferably other genes, may be indicative of a marker or " Quot; read out ".

(E. G., A compound, drug or small molecule) that modulates PD-L3 or VISTA activity (e. G., Identified in the screening test described herein) A gene containing PD-L3 or VISTA, which is regulated in the cell, can be identified. Thus, in order to study the effect of the formulation on PD-L3 or VISTA-related disorders, for example in clinical trials, cells can be isolated and PD-L3 or VISTA and RNA preparations And can be analyzed. The level of gene expression (e.g., gene expression pattern) can be measured by Northern blot analysis or RT-PCR as described herein, or by measuring the amount of the produced polypeptide, as one of the methods described herein, Or by measuring the activity levels of VISTA or other genes. In this way, the gene expression pattern serves as a marker, marking the physiological response of the cell to the agent. Thus, such a response state can be determined at various times before, and during, the treatment of the agent with the individual. In a preferred embodiment, the present invention provides a treatment effect of an agent (e.g., an agent, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening test described herein) Comprising the steps of: (i) obtaining a pre-administration sample from an individual prior to administration of said preparation; (ii) detecting the level of expression of the PD-L3 or VISTA polypeptide, mRNA or genomic DNA in the battle sample; (iii) obtaining one or more post-treatment samples from the subject; (iv) detecting the level of expression or activity of the PD-L3 or VISTA polypeptide, mRNA or genomic DNA in the treated sample; (v) comparing PD-L3 or VISTA polypeptide, mRNA or genomic DNA expression or activity levels in the pretreatment sample to PD-L3 or VISTA polypeptide, mRNA or genomic DNA in the sample after treatment; And (vi) thus varying the administration of the agent from the subject. For example, increased administration of the agent is desirable to increase PD-L3 or VISTA expression and activity, i.e. increase the effectiveness of the agent, to a higher level than that detected. Alternatively, a reduced administration of the agent is desirable to decrease the expression or activity of PD-L3 or VISTA for a level lower than that detected. According to this example, PD-L3 or VISTA expression or activity can be used as an indicator of drug efficacy, even in the absence of observable phenotypic responses.

D. Methods of Treatment

The present invention relates to a method for the treatment of a disorder characterized by the production of a PD-L3 or VISTA protein in the form of PD-L3 or VISTA protein, or a PD-L3 or VISTA protein form having reduced or excessive production, Both prophylactic and therapeutic methods for treating a subject at risk (or vulnerable). The PD-L3 or VISTA antibodies of the present invention also detect and isolate PD-L3 or VISTA proteins, for example by modulating the interaction of PD-L3 or VISTA with its corresponding receptor, Or to modulate the bioavailability of the VISTA protein, and to modulate PD-L3 or VISTA activity.

1. Prophylactic Methods

In one aspect, the invention provides a method for treating abnormal or unwanted PD-L3 or VISTA expression in a subject by administering to the subject a PD-L3 or VISTA polypeptide or agent that modulates PD-L3 or VISTA expression or at least one PD-L3 or VISTA activity, L3 or VISTA expression or activity. Individuals at risk for a disease or disorder that is caused or produced by abnormal or unwanted PD-L3 or VISTA expression or activity may be identified by one or a combination of, for example, diagnostic or prospective tests described herein . A prophylactic agent that prevents or slows the progress of the disease or disorder may be administered prior to the occurrence of the characteristic symptoms of PD-L3 or VISTA predisposition. For example, PD-L3 or VISTA polypeptide PD-L3 or VISTA agonists or PD-L3 or VISTA antagonists (e.g., anti-PD-L3 or VISTA antibodies) preparations, depending on the PD- Can be used to treat the subject. The appropriate formulation can be determined based on the screening test described herein.

2. Therapeutic methods

An important aspect of the invention includes methods of modulating PD-L3 or VISTA expression or activity, or interaction with its natural binding partner. In relation to therapy, PD-L3 or VISTA inhibits CD28 co-stimulation, inhibits TCR activity of immune cells, inhibits the proliferation of activated immune cells (CD4 + and CD8 + T cells) (IL-2, gamma interferon) and to transmit inhibitory signals to immune cells. Thus, the activity and / or expression of PD-L3 or VISTA, as well as the mutual binding between PD-L3 or its binding partner in VISTA and T cells, can be modulated to regulate the immune response. Since PD-L3 or VISTA binds to inhibitory receptors (of T cells), upregulation of PD-L3 or VISTA activity should result in downregulation of the immune response whereas downregulation of PD-L3 or VISTA activity Immunomodulation should result in upregulation. In a preferred embodiment, PD-L3 or VISTA binds to an inhibitory receptor. As opposed to intuitive PD-L3 or VISTA-Ig fusion proteins produced by attempting to enhance the inhibitory activity of PD-L3 or VISTA-Ig fusion proteins in vitro (in the presence of PD-L3 or VISTA-Ig) The L3 or VISTA specific antibodies reverse the expected action in vivo. That is, the antigens are found immunosuppressively in vivo.

The artificial method of the present invention modulates the expression or activity of a PD-L3 or VISTA polypeptide or an agent that modulates one or more of the PD-L3 or VISTA activities associated with the cell, such as PD-L3 or VISTA, and / Or PD-L3 or VISTA and a natural binding partner thereof. PD-L3 or VISTA antibody, a PD-L3 or VISTA agonist or antagonist, a PD or a polypeptide or a naturally occurring binding partner of PD-L3 or VISTA, PD-L3 or VISTA antibody, Such as peptidomimetics, PD-L3 or VISTA peptidomimetics, or other small molecules of the L3 or VISTA agonist or antagonist. In addition, soluble forms of PD-L3 or VISTA can be used to mediate binding of PD-L3 or VISTA and any of its natural binding partners or ligands.

An agent that modulates PD-L3 or VISTA expression is, for example, a recombinant vector for the expression of antisense nucleic acid molecules, triplex oligonucleotides, ribozymes, or PD-L3 or VISTA polypeptides. For example, oligonucleotides complementary to the PD-L3 or VISTA polypeptide translation initiation site may be synthesized. One or more antisense oligonucleotides may be added to the cell culture medium, generally at 200 mug / ml, or injected into a patient to prevent the synthesis of PD-L3 or VISTA polypeptides. The antisense oligonucleotide is absorbed into cells and hybridized with PD-L3 or VISTA mRNA to prevent translation. Alternatively, an oligonucleotide that binds double-stranded DNA can be used to form a triple structure to prevent DNA annealing or transcription. In any case, as a result, the synthesis of PD-L3 or VISTA polypeptides is blocked. When PD-L3 or VISTA expression is regulated, preferably the modulation occurs by other means than knocking out the PD-L3 or VISTA gene.

By virtue of the fact that they regulate the amount of PD-L3 or VISTA in the cell, the agent that regulates its expression also regulates the total amount of PD-L3 or VISTA activity in the cell. In some embodiments, the agent that modulates PD-L3 or VISTA stimulates one or more PD-L3 or VISTA activities. Examples of such irritant preparations include active PD-L3 or VISTA polypeptides and nucleic acid molecules encoding PD-L3 or VISTA introduced into cells. In yet another embodiment, the agent inhibits one or more of PD-L3 or VISTA activity. Examples of such inhibitory agents include antisense PD-L3 or VISTA nucleic acid molecules, anti-PD-L3 or VISTA antibodies, PD-L3 or VISTA inhibitors and compounds identified in said individual screening assays. In another preferred embodiment, the inhibitory agent is a combination of an anti-PD-L3 or VISTA antibody and an anti-PD-L1 or anti-PD-L2 antibody. The modulatory method may be carried out in vitro (e.g., by contacting the cell with the agent) or by contacting the agent with the cell (e. G., By administering the agent to the subject) in vivo. As noted above, the present invention is directed to the use of a PD-L3 or VISTA polypeptide or nucleic acid molecule for the manufacture of a medicament for the treatment or prophylaxis of diseases or disorders which may benefit from upregulating or downregulating PD-L3 or VISTA polypeptides, for example unwanted, The invention provides a method of treating an individual having a disease characterized by activity. In certain embodiments, the methods involve administering a formulation (e.g., a formulation identified by a screening test described herein) or a combination of agents that modulate PD-L3 or VISTA expression or activity. In another embodiment, the method involves administering a PD-L3 or VISTA polypeptide or nucleic acid molecule as a therapeutic agent to compensate for reduced, abnormal or unwanted PD-L3 or VISTA expression or activity.

Diseases that can be treated using PD-L3 or VISTA binding agents of an individual have been previously identified and include a variety of inflammatory, autoimmune, cancer, allergic, and infectious diseases. A particular preferred indicator is multiple sclerosis.

Stimulation of PD-L3 or VISTA activity is desirable when PD-L3 or VISTA is abnormally downregulated and / or when increased PD-L3 or VISTA activity has a beneficial effect. Likewise, inhibition of PD-L3 or VISTA activity is desirable when PD-L3 or VISTA is abnormally up-regulated and / or when reduced PD-L3 or VISTA activity has a beneficial effect. Examples of formulations for down-regulating PD-L3 or VISTA include antibodies (PD-L3 or VISTA antagonists), such as antisense nucleic acid molecules, PD-L3 or VISTA, PD-L3 or VISTA And antibodies that recognize and block PD-L3 or VISTA-responsive receptors, and compounds that block the interaction of PD-L3 or VISTA with its naturally occurring binding partner in immune cells For example, a soluble, monovalent PD-L3 or VISTA molecule; a soluble form of PD-L3 or VISTA that does not bind to an Fc receptor in an antigen presenting cell; and a compound identified in the screening test of the individual) do. Exemplary agents for up-regulating PD-L3 or VISTA include (i. E. PD-L3 or VISTA agonists), for example nucleic acid molecules encoding PD-L3 or VISTA polypeptides, PD-L3 or VISTA unit forms, A compound that increases the expression of PD-L3 or VISTA, a compound that enhances the interaction of PD-L3 or VISTA with its naturally occurring binding partner, and cells expressing PD-L3 or VISTA.

3. Downregulation of the immune response

There are many embodiments of the present invention to down-regulate the immune response by upregulating the inhibitory function of PD-L3 or VISTA polypeptides. Downregulation may be in the form of inhibiting or blocking an already progressing immune response, or may involve blocking the induction of an immune response. The function of activated immune cells can be inhibited by down-regulating the immune cell response or by inducing specific anergy of immune cells, or both. For example, in some embodiments, where PD-L3 or VISTA binds to a repressive receptor, a form of PD-L3 or VISTA that binds to the inhibitory receptor, such as a multivalent PD -L3 or VISTA can downregulate the immune response. In certain embodiments of the invention, the activated antibody used to stimulate PD-L3 or VISTA activity is bispecific. For example, the antibody includes a PD-L3 or VISTA binding site and another binding site that targets a cell surface receptor in an immune cell, such as a T cell, a B cell, or a myeloid cell. In yet another embodiment, the antigens comprise a PD-L3 or VISTA binding site, as well as those that bind to a B cell antigen receptor, a T cell antigen receptor, or an FC receptor to target a molecule for a specific cell population Binding sites may be included. Screening of secondary antibodies against bispecific antibodies provides flexibility in the selection of cell populations to be targeted for inhibition. Agents that promote PD-L3 or VISTA activity and enhance the cross-linking of PD-L3 or VISTA and its natural binding partner (e.g., PD-L3 or VISTA activating antibody or PD-L3 or VISTA activated small) Their ability to inhibit their immune cell proliferation and / or inhibit the functional mechanism, or to induce the host when added in vitro. For example, the cells may be cultured in the presence of agents that stimulate signal transduction through the activation receptor. A number of art-recognized readouts of cellular activation are applied in the presence of activating agents, for example, to measure cell proliferation or functional mechanism (e.g., antibody production, cytokine production, macrophage action) . The ability of a test agent to block such activation can be readily determined by measuring the ability of the agent to exhibit the effect of reducing the proliferation or functional group to be measured. In some embodiments, at low antigen concentrations, PD-L3 or VISTA immune cell interactions inhibit the potent B7-CD28 signal. In another embodiment, at high antigen concentrations, PD-L3 or VISTA immune cell interactions may reduce cytokine production but do not inhibit T cell proliferation. Thus, the ability to block activation of the test compound can be determined by measuring cytokine production and / or proliferation at different concentrations of the antigen.

In certain embodiments of the invention, resistance may be induced to specific antigens by co-administration of the antigen with a PD-L3 or VISTA agonist. For example, resistance can be induced to specific polypeptides. In some embodiments, the immune response to an allergen or an external polypeptide for which an immune response is undesirable can be inhibited. For example, patients receiving factor VIII often produce antibodies to these blood clotting factors. Co-administration of an agent that stimulates PD-L3 or VISTA activity or an interaction with a natural binding partner thereof (or, for example, by physically linking PD-L3 or VISTA to Factor VIII by cross-linking, ) May result in downregulation of the immune response.

In some embodiments, the PD-L3 or VISTA agonist, and another agent capable of blocking the activity of the co-stimulatory receptor in the immune cell, can be used to down regulate the immune response. Exemplary molecules include: an agonist form of another PD ligand, a soluble form of CTLA-4, an anti-B7-1 antibody, an anti-B7-2 antibody, or a combination thereof. Alternatively, a combination of two separate peptides (e.g., PD-L3 or VISTA polypeptide with a blocking hive of B7-2 and / or B7-1 polypeptide) or antibody (anti-B7-2 and / or anti- B7.1 blocker monoclonal antibody, an activating antibody to a PD-L3 or VISTA polypeptide) can be combined as a single composition, or administered separately, to downregulate an immune cell mediated immune response in an individual (Concurrently or sequentially). In addition, the therapeutically active amount of one or more peptides having PD-L3 or VISTA polypeptide activity, in combination with one or more polypeptides having B7-1 and / or B7-2 activity, May be used in combination with an adjusting reagent. Examples of other immunoregulatory agents include, but are not limited to, a co-stimulatory signal (e.g., for CD28 or ICOS), an antibody that activates a repressive signal via CTLA4 and / or other immune cell markers (e.g., CD40, CD40 ligand, Fusion proteins (e.g., CTLS4-Fc or PD-2-Fc) and immunosuppressive drugs (such as rapamycin, cyclosporin A, or FK506) . In addition, PD-L3 or VISTA polypeptides may be useful in the production of therapeutic agents that block immune cell function by cell destruction. For example, a portion of PD-L3 or VISTA may be associated with a toxin to produce a cytotoxic agent that may cause destruction of the cell it binds to.

For cytotoxic agents, the polypeptides of the present invention may be linked or operatively associated with toxins using techniques known in the art. A variety of toxins that are capable of binding to the polypeptides or antibodies of the invention are known. Examples include many useful plant-, fungal-, or bacterial-derived toxins, examples of which include: various A chain toxins, particularly the ricin A chain; Ribosomes inactivating proteins such as saporin or gelonin; Alpha-sarcin; Aspergillin; Restictosin; And ribonuclease, such as placental nuclease, angiogenic, diphtheria toxin, or pseudomonas endotoxin. A preferred toxin moiety for use in conjunction with the present invention is a deglycosylated A chain (U.S. Pat. No. 5,776,427) which is a toxin A chain that has been modified such that the carbohydrate moiety is modified or removed.

Administration with one or a combination of the above cytotoxic agents (e.g., in combination with PD-L3 or VISTA lysine (alone or in combination with PD-Ll-lysine) may result in the death of immune cells, particularly PD- Dependent mechanism because PD-1 is derived from the surface of an activated lymphocyte, or by PD-L3 or VISTA < RTI ID = 0.0 > PD-L3 or VISTA polypeptides by the removal of a polypeptide and a cytotoxic drug such as lysine, saponin or calicheamicin may be used to target the depletion of the specific cell. In another embodiment, the toxin may be conjugated to an anti-PD-L3 or VISTA antibody to target the death of PD-L3 or VISTA-expressing antigen presenting cells. In yet another embodiment, the PD-L3 or VISTA-antigen-toxin is a double-specific Such a bispecific antibody can be detected by using a marker specifically expressed in, for example, a specific kind of cell, for example, B lymphocytes, monocytes, dendritic cells or Langerhans cells (And thus stimulate the voice signal function of PD-L3 or VISTA) by activating PD-L3 or VISTA activity or PD-L3 or VISTA-immune cell interactions Modulating is useful for down-regulating the immune response in autoimmune diseases such as graft-versus-host disease (GVHD) or allergy, or systemic lupus erythematosus and multiple sclerosis, for example in tissue, skin and organ transplantation situations. For example, blocking of immune cell function results in reduced tissue destruction in tissue grafts. Typically, in tissue grafts, PD-L3 or VISTA, or its natural binding partner (e.g., PD-L3 or VISTA) in immune cells, is initiated by an immune response that recognizes the cell as a foreign entity, Administration of a molecule that promotes the interaction of the L3 or VISTA polypeptide with a molecule that promotes the interaction of the molecule, prior to or at the time of transplantation, alone or in combination with other down-regulating agents, may inhibit the formation of a co-molecular signal. In addition, the promotion of PD-L3 or VISTA activity is sufficient to energize immune cells, and thus may cause tolerance in an individual.

In order to achieve sufficient immunosuppression or tolerance in an individual, it is desirable to block the function of co-stimulation of other molecules. For example, prior to or at the time of transplantation, a soluble form of a peptide combination having the activity of each of the antigens, or B7-1 and / or B7-1, by administering a blocking antibody to the antigens (either separately or in a single composition) It is desirable to block the function of B7-2. Alternatively, it is desirable to promote the inhibitory activity of PD-L3 or VISTA and inhibit the co-stimulatory activity of B7-1 and / or B7-2. Other downregulatory agents that may be used in connection with the down-regulating method of the present invention include, for example, agents that deliver an inhibitory signal through CTLA4, a soluble form of CTLA4, an antibody that activates an inhibitory signal via CTLA4, Blocking antibodies to other immune cell markers, or soluble forms of other receptor ligand pairs (such as agents that destroy the interaction between CD30 and CD40 ligands, such as anti-CD40 ligand antibodies), antibodies to cytokines , Or an immunosuppressive drug. For example, activating the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA with its natural binding partners is useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of immune cells that respond to self tissue and promote cytokine production, and of an autoantibody associated with the pathology of the disease. Blocking activation of self-reactive immune cells can reduce or eliminate disease symptoms. Administration of PD-L3 or VISTA, or the administration of an agent that promotes the interaction of PD-L3 or VISTA with its natural binding partner, may result in prolonged release from the disease, the antigen-specific immunity of autoreactive immune cells Lt; / RTI > In addition, co-administration of an agent that blocks the co-stimulation of immune cells by destroying the receptor-ligand interaction of the B7 molecule with the co-stimulatory receptor will block the production of self-reactive antibodies or cytokines that may be associated with the disease progression May be useful for inhibiting the activation of immune cells. The effect of a reagent that prevents or mitigates autoimmune disorders can be determined using many well-characterized animal models of human autoimmune disease. Examples include experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, diabetes mellitus in rat autoimmune collagen arthritis, NOD mice and BB rats, and mouse experimental myasthenia gravis experimental gravis myasthenia) (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Inhibition of immune cell activation is useful, for example, in the treatment of allergic and allergic reactions by inhibiting IgE production. Agents that promote PD-L3 or VISTA activity or interaction with PD-L3 or VISTA and its natural binding partner may be administered as an allergic entity to inhibit immune cell-allergic response in an individual. The stimulation of interaction with PD-L3 or VISTA activity or its natural binding partners may be accompanied by exposure to allergens linked to appropriate MHC molecules. The allergic response may be systemic in nature or local, depending on the pattern of IgE accumulation of mast cells or basophils and the administration of allergen. Thus, the immune cell-mediated allergic response may be inhibited by systemically or topically administering an agent that promotes PD-L3 or VISTA activity or PD-L3 or VISTA-immune cell interaction.

Inhibition of immune cell activation through interaction with PD-L3 or VISTA activity or with PD-L3 or VISTA and its natural binding partners is a therapeutic (e.g., by virus or bacteria) infection in pathological infections of immune cells . ≪ / RTI > For example, in acquired immunodeficiency syndrome (AIDS), viral replication can be stimulated by immune cell activation. Stimulation of PD-L3 or VISTA activation can result in inhibition of viral replication, thus alleviating the progression of AIDS.

In addition, down-regulation of the immune response through stimulation of the interaction of PD-L3 or VISTA activity or PD-L3 or its natural binding partner with VISTA may be useful for treating autoimmune attack of self-organization. Thus, diseases (e.g., heart disease, myocardial infarction, or atherosclerosis) caused or aggravated by autoimmune attack increase PD-L3 or VISTA activity or the binding of PD-L3 or VISTA and its natural binding partner Or can be improved. Thus, by stimulating the interaction of PD-L3 or VISTA activity or PD-L3 or its corresponding receptor with VISTA, an autoimmune attack, such as autoimmune disorders (as well as heart disease, myocardial infarction or atherosclerosis) It is within the scope of the present invention to control disease that is exacerbated by the disease.

4. Upregulation of the immune response

As a means of upregulating the immune response, inhibition of the interaction of PD-L3 or VISTA activity or PD-L3 or its natural binding partners with VISTA is useful in therapy. Upregulation of the immune response may be in the form of enhancing the existing immune response or inducing an initial immune response. For example, the promotion of immune response through inhibiting PD-L3 or VISTA activity is useful in the case of microorganisms, such as bacterial, viral or parasitic infections, or in the case of immunosuppression. For example, in some embodiments, a soluble form of PD-L3 or a non-active antibody (i. E., Blocking antibody) or a PD-L3 or VISTA to VISTA activity inhibiting agent such as PD-L3 or VISTA Suppressive agents are therapeutically useful in situations where up-regulation of antibodies and cell-mediated responses is beneficial, resulting in a more rapid or total clearing of viruses, bacteria, or parasites. Such diseases include viral skin diseases, such as herpes or shingles, wherein the agent is delivered locally to the skin. In addition, systemic viral diseases such as influenza, general cold and encephalomyelitis can be alleviated by systemically administering the agent. In certain instances, it may be desirable to administer other agents that upregulate the immune response to further enhance the immune response, e.g., a form of a B7 system member that delivers a signal through a co-stimulatory receptor.

Alternatively, the immune cells are removed from the patient and the immune cells in vitro are contacted with an agent that inhibits PD-L3 or VISTA activity or interaction with PD-L3 or VISTA and its natural binding partner, and in vitro By re-introducing the stimulated immune cells into the patient, the immune response can be enhanced within the infected patient. In another embodiment, a method of enhancing an immune response comprises isolating infected cells from a patient, e. G., Virus-infected cells, and allowing the cells to express all or part of the PD-L3 or VISTA molecule on their surface Transfecting them with nucleic acid molecules encoding a form of PD-L3 or VISTA that can not be combined with a binding partner, and reintroducing said transduced cells into said patient. The transduced cells may be able to prevent inhibitory signals and thus may activate immune cells in vivo.

Agents that inhibit the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA and its natural binding partner can be used prophylactically in vaccines against polypeptides derived from a variety of polypeptides, e. G., Pathogens. Immunity to the pathogen, e. G., The virus, may be induced by vaccination with a viral polypeptide with an agent that inhibits the activity of PD-L3 or VISTA. Alternatively, vectors comprising genes encoding both pathogenic antigens and forms of PD-L3 or VISTA that block the interaction of immune cells with PD-L3 or VISTA can be used for vaccination. Nucleic acid vaccines can be administered in a variety of ways, for example, by infusion (e.g., intramuscular, intradermal or DNA-coated gold particles percutaneously, Biolistic injection (Haynes et al. (1996) J. Biotechnol. 44:37), which is injected with a particle promoter or a gene gun using a compressed gas in order to achieve the desired effect. Alternatively, the nucleic acid vaccine may be administered by non-invasive means. For example, pure or lipid-formulated DNA can be delivered to a target site, for example, Peyers patches, via oral delivery of the respiratory system or DNA (Schubbert (1997) Proc Natl. Acad. Sci. USA 94: 961). Attenuated microorganisms can be used to deliver to the surface of the spots (Sizemore et al. (1995) Science 270: 29).

In another embodiment, the antigen of the vaccine is a self-antigen. The vaccine is useful for modulating tolerance in an organism. Immunization with self-antigens and agents that block the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA and its natural binding partner may break tolerance (ie, interfere with the resistance of the self antigens). In addition, the vaccine may comprise a reinforcing agent such as alum or a cytokine (e.g., GM-SCF, IL-12, B7-1 or B7-2). In certain embodiments, agents that inhibit the interaction of PD-L3 or VISTA activity or PD-L3 or a natural binding partner thereof with VISTA to provide immunity from infection include, for example, PD-L3 or VISTA polypeptides Or blocking antibodies, and MHC class I. Alpha. May be administered with MHC class I polypeptides by cells transduced to co-express the chimeric polypeptide and beta 2 microglobulin. For example, the viral pathogens used in vaccines include: hepatitis B virus, hepatitis C virus, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria, and schistosomiasis.

In another aspect, inhibition of the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA and its natural binding partner may be useful in the treatment of tumor immunity. Tumor cells (sarcoma, sarcoma, lymphoma, leukemia, nephrocytomas or adenocarcinomas) can be transduced with nucleic acid molecules that inhibit PD-L3 or VISTA activity. Such a molecule may be, for example, a nucleic acid molecule that is antisense to PD-L3 or VISTA, or may encode a non-active anti-PD-L3 or VISTA antibody. In addition, the molecule may be a variable region of an anti-PD-L3 or VISTA antibody. Preferably, the tumor cells may also be introduced with other polypeptides that activate co-stimulation (e. G., B7-1 or B7-2). The transduced tumor cells are returned to the patient, which results in the inhibition of PD-L3 or VISTA activity. Alternatively, gene therapy techniques can be used to target tumor cells for transduction in vivo.

In addition, the stimulation of the immune response to the tumor cells can be induced by PD-L3 or VISTA activity or PD-L3 or VISTA activity by inhibiting the interaction of PD-L3 or VISTA with its natural binding partner ≪ / RTI > by inhibiting the natural binding partner of < RTI ID = 0.0 > Preferred examples of such formulations include antibodies that recognize and block antisense nucleic acid molecules, PD-L3 or VISTA, and compounds that block the interaction of PD-L3 or VISTA with its naturally occurring binding partner in immune cells For example, soluble forms of PD-L3 or VISTA molecules that do not bind Fc receptors in antigen-presenting cells; soluble forms of PD-L3 or VISTA binding partners; and compounds identified in said individual screening assays. In addition, tumor cells lacking MHC class I or MHC class II molecules or failing to express sufficient amounts of MHC class I or MHC class II molecules can be classified as MHC class I alpha. Chain polypeptides and beta 2 microglobulin polypeptide or MHC class II. Alpha. Chain polypeptides and MHC class II .beta. May be transduced with a nucleic acid encoding all or part of the chain polypeptide (e. G., A cytoplasmic-domain cleavage moiety) and thus may express MHC class I or MHC class II polypeptides on the cell surface. Expression of the appropriate MHC class I or class II together with a PD-L3 or VISTA inhibitory polypeptide or antisense nucleic acid induces a T cell mediated immune response to the transfected tumor cells. Alternatively, in order to promote the presentation of tumor-associated antigens and induce tumor-specific immunity, genes encoding MHC class II-related polypeptides, such as antisense constructs that block expression of an invariant chain, may also be PD-L3 or VISTA inhibitors Or co-transduced with DNA encoding a polypeptide or antisense nucleic acid. The expression of B7-1 by B7-negative mouse tumor cells has been shown to induce T cell mediated specific immunity with delayed protection against tumor rejection and tumor transplantation in mice (Chen, L. et al. (1992 Nascar, S. et al. (1993) Proc Natl. Acad. Sci., 90: 5687-5690). Thus, induction of an immune cell-mediated immune response in a human individual may be sufficient to overcome tumor-specific resistance in an individual. In yet another embodiment, the immune response can be stimulated by inhibiting the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA with its natural binding partner, and therefore the resistance present first can be overcome . For example, an immune response to an antigen for which an individual does not develop a significant immune response, for example, a tumor-specific antigen, has the ability to bind PD-L3 or VISTA activity or its natural binding partner of PD-L3 or VISTA , And can be used as augmenting agent to enhance the response to external antigens in the course of active immunization.

In certain embodiments, an immune cell is obtained from an individual to expand said population of immune cells and is administered in vitro with a PD-L3 or VISTA activity or an agent that inhibits the interaction of PD-L3 or VISTA with its natural binding partner ≪ / RTI > In yet another embodiment, the immune cell is then infused into the subject. The immune cells may be stimulated to proliferate in vitro, for example, by providing the immune cells with primary activation signals and co-stimulation signals known in the art. Various forms of PD-L3 or VISTA polypeptides and agents that inhibit PD-L3 or VISTA activity can also be used to co-stimulate the proliferation of immune cells. In certain embodiments, the immune cells are selected from the group consisting of PCT application no. And cultured in vitro according to the method described in WO94 / 29436. The co-stimulating molecule is either soluble, bound to a cell membrane, or bound to a solid surface, such as a bead.

In another embodiment, in performing any of the methods described herein, it is within the scope of the present invention to upregulate the immune response by administering one or more agents. For example, the use of stimulant forms of other molecules known to stimulate the immune response, such as cytokines, adjuvants, or co-stimulatory molecules, or their ligands, may stimulate PD-L3 or VISTA activity or PD-L3 or VISTA and its Can be used with agents that inhibit the interaction of natural binding partners.

E. PD - L3  or VISTA  Active or PD - L3  or VISTA And the regulation of the interaction of its counterpart receptors in T cells Love  Identification of Modified Cytokines

The PD-L3 or VISTA molecule described above is capable of enhancing or inhibiting its production in immune cells against the modulation of the produced cytokine, or the interaction of PD-L3 or VISTA activity or PD-L3 or its natural binding partner with VISTA RTI ID = 0.0 > cytokine < / RTI > The immune cells may be optionally stimulated in vitro with a primary activation signal, for example a T cell may be an antigen coupled to a pharbol ester, an anti-CD3 antibody, or preferably an MHC class II molecule, and a given For example, by stimulating a co-stimulatory signal, e.g., a stimulatory form of a B7 antigen, such as by transfecting the surface with a nucleic acid encoding a B7 polypeptide and expressing the peptide on its surface, or by the soluble, It can be stimulated. The cells can then be contacted with cells expressing PD-L3 or VISTA (e.g., antibodies against PD-L3 or VISTA). Known cytokines secreted into the medium can be identified by ELISA or by the ability of the antibody to block cytokines that inhibit the proliferation of immune cells or the proliferation of other cytokines induced by the cytokines. For example, IL-4 ELISA kits, such as IL-7 blocking antibodies, are available from Genzyme (Cambridge, Mass.). Blocking antibodies to IL-9 and IL-12 are available from the Genetics Institute (Cambridge, Mass.). In the cytokine profile, the effect of stimulating or blocking the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA and its binding partner can then be determined. PD-L3 or VISTA clearly suppresses the expression of IL-2 and gamma interferon by immune cells, as described above and in the Examples.

In addition, the above-described in vitro immune cell co-stimulation test can be used in a method for identifying a novel cytokine that can be regulated by modulating PD-L3 or VISTA activity. For example, stimulation of the ICOS pathway appears to enhance the secretion of IL-10, where stimulation of the CD28 / CTLA4 pathway appears to increase IL-2 secretion (Hutloff et al. (1999) Nature 397: 263 ). If the specific activity induced by co-stimulation, e. G., Immune cell proliferation, can not be inhibited by adding a blocking antibody to a known cytokine, then the activity will result from the action of an unknown cytokine. After co-stimulation, such cytokines can be purified from the medium by a common method, and its activity can be measured by its ability to induce immune cell proliferation.

In order to identify cytokines that may play an important role in the induction of resistance, the in vitro T cell co-stimulation assay described above may be used. In this case, the T cell may be a given primary activation signal and may be contacted with a selected cytokine, but not a given co-stimulatory signal. If the immune cells do not respond (e. G., Proliferation or cytokine production) they will become resistant, and the cytokine will not prevent the induction of resistance. However, when the immune cells react, induction of resistance is prevented by the cytokine. The cytokine capable of inducing tolerance is a more effective means of inducing resistance in recipients or individuals with autoimmune diseases, together with a reagent that blocks B lymphocyte antigens. Can be targeted in vivo. For example, a cytokine blocking antibody may be injected into a subject along with an agent that promotes the interaction of PD-L3 or VISTA activity or PD-L3 or VISTA with the binding partner.

Thus, in summary, a new member of the Programmed Death Ligand (PDL) system has been identified, which is expressed in Treg cells. The novel protein was named PD-L3 or VISTA. The PD-L family of receptors is a class I transmembrane protein containing a single IgV domain, while the ligand is a class I transmembrane protein expressing IgV and IgC extracellular domains. PD-L3 or VISTA co-stimulates the proliferation of T cells in vitro by the end of the PDL system. In addition, the expression of PD-L3 or VISTA increases αCD3-activated Treg cells and decreases in the presence of αGITR.

Second, TNF-like, protein was identified as upregulated by αCD3 / αGITR stimulation. This protein is named Treg-sTNF. The protein may be associated with contact-dependent and immunological paracrine inhibition and is therefore useful in diseases and disorders related to modulation (up or down) of the immune response and Treg signaling. For example, the PD-L3 or VISTA protein can be used as a co-stimulatory signal to stimulate or enhance immune cell activation. PD-L3 or VISTA proteins and PD-L3 or VISTA binding agents and PD-L3 or VISTA agonists and antagonists are particularly useful for treating immune disorders, wherein modulation of T cell immunity, such as activation, differentiation and / Modulation of proliferation is preferred, and modulation of T cells during, inter alia, interactions between CD4 + T and CD8 + T cell proliferation, cytokine production, and T cells and myeloid derived APCs is desirable.

The present invention is further described by the following examples, which are not to be construed as limitations of the present invention. The contents of all references, patents, and published patent applications cited throughout this application, as well as maps and sequence listings, are incorporated herein by reference.

Example

The following materials and methods were used in the examples according to materials and methods.

Expression profile  ( expression profiling )

In order to facilitate comparison of the established expression profiles of Treg cells, normal growth and activation conditions were applied (McHugh, et al. (2002)). Briefly, freshly isolated Treg cells (~ 96% positive) were incubated with 10% fetal bovine serum in a 24-well plate precoated with anti-CD3, with or without anti-GITR (Shimizu, et al. (2002)) at a concentration of 106 / ml in complete RPMI medium with fetal bovine serum and 100 units of IL-2. The cells were incubated at 37 ° C for 0 and 12 hours and the RNA was purified and then analyzed using the Affymetirx® mouse genome A430 oligonucleotide array.

By comparing the above data from resting or activated CD4 + CD25 + T cell populations, the gene expression profile can be determined by one of the methods established in the art (Gavin, et al. (2002); McHugh, et al (2002) . ≪ / RTI > In order to identify genes that are regulated by GIRT signaling, gene expression profiles were compared between different cell populations with or without anti-GITR treatment. We made a list of known as well as unknown genes, including PD-L3 or VISTA and Treg-sTNF, which were not previously characterized.

mouse

C57BL / 6 mice, and OTII CD4 transgenic mice were purchased from Jackson Laboratory. FoxP3-GFP receptor mice are as described previously (Frontenor, J. D., Rasmussen, J. Williams, LM, Dooley, J. L., Farr, A. G., and Rudensky, A. Y. (2005)). Regulatory T cell lineage specification by the forkhead transcription factor foxp3, Immunity 22, 329-341) and was kindly provided by Alexander Rudensky University of Washington School of Medicine Seatle, WA. PD-l KO mice were treated with (1999) Development of lupus-like autoimmune diseases by Nishimura, H., Nose, M., Hiai, H., Minato, N., Honjo, T. Nashimura, H., Okazaki, T., Tanaka, Y., Nakatani, L., Hara, M., Matsumori, A, Immunity 11, 141-151; Disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. (2001) Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice, Science 291, 319-322). . All animals were housed in a pathogen-fee facility at Dartmouth Medical School.

Abs , Cell lines and reagents:

The antibodies αCD3 (2C11), αCD28 (PV-1), αCD4 (GK1.5), αCD8 (53-6.7), αCD11b (M1 / 70), αF4 / 80 (BM8), αCD11c (RB6-8C5), alpha PD-L1 (MIN5), alpha PD-L2 (TY25), alpha B7-H3 (M3.2D7) and alpha B7-H4 (188) were purchased from Ebioscience. LPS (Sigma), recombinant mouse IFN (Peprotech), human IL-2 (Peprotech) and soluble PD-L1-Ig fusion protein (R & D system) were used at the indicated concentrations. Complete Freund's adjuvant (CFA) and chicken ovalbumin (OVA) were purchased from Sigma. The CHO cell line expressing the MHC II molecule I-Ad and the co-stimulatory molecule B7-2 was obtained from Dr. It was kindly provided by Arlene Sharpe (Harvard Medical School).

PD - L3  or VISTA Molecule Cloning , Retroviral production and retroviral transduction of cells

The full length of PD-L3 or VISTA was cloned from isolated mouse CD4 + T cells. Total RNA was isolated from CD4 + T cells using a Qiagen RNA mini kit. Using the Bio-Rad iScriptTM cDNA synthesis kit, cDNA was produced. PD-L3 or VISTA was amplified and cloned into the EcorI-XhoI site of the retroviral vector pMSCV-IRES-GFP (Zhang, X., and Ren, R. (1998). Bcr-Abl efficiently induces Bllod 92, 3928-3840), in which the IRES-GFP fragment was replaced by RFP, and thus, the IRES-GFP fragment was replaced with the < RTI ID = 0.0 & A fusion protein of PD-L3 or VISTA in which RFP was fused at the N-terminus was made. Helper-free retrovirus was isolated from HEK293T by transient transduction of the PD-L3 or VISTA-RFP retroviral vector with the ecotrophic package vector pCL-Eco (IMGENEX corp.) Was produced. Retroviral transduction of mouse T cell line EL4 cells or bone marrow-derived DCs was performed by spin infection at 2000 RT for 45 min in the presence of 8 ug / ml polybrene (Sigma).

PD - L3  or VISTA - Ig  Production of fusion protein

The extracellular domain of PD-L3 or VISTA (amino acids 32-190) was amplified and cloned into the SpeI-BamHI site of the parental vector CDM7B (Hollenbaugh, D., Douthwright, J., McDanald, Aruffo, A. (1995) J Immunol Methods 188, 1-7). These vectors contain a constant and hinge region mutant form of human IgG1, which has much reduced binding to Fc receptors. The resulting vector CDM7B-PD-L3 or VISTA is cotransfected with the CHO (dhfr-) cell line (ATCC # CRL-9096) with the DHFR expression vector pSV-dhfr (McIvor, RS, and Simonsen, CC 1990) Nucleic Acid Res 18, 7025-7032). Stable CHO cell lines expressing PD-L3 or VISTA-Ig are selected in the medium MEM-alpha (Invitrogen) without nucleotides. Further, amplification with 0.5-1 mu methotrexate (Sigma M9929) provides clones that express soluble PD-L3 or VISTA-Ig fusion proteins at high levels. Further, the fusion protein was purified from the medium using general protein-G column affinity chromatography.

PD - L3  or VISTA  Production of monoclonal antibodies

The armenian hamster was immunized 4 times weekly with EL4 cells overexpressing PD-L3 or VISTA and then boosted with PD-L3 or VISTA-Ig fusion protein emulsified in CFA. Hamster splenic cells were obtained using common hybridoma fusion techniques and fused with the myeloid cell line SP2 / 0-Ag14 (ATCC # CRL-1581) 4 days after the above-mentioned induction (Shulman, M., Wilde, CD, and Kohler , G. (1978), A better cell for making hybridomas, secreting specific antibodues, Nature 276, 269-270). Hybridoma clones secreting PD-L3 or VISTA specific antibodies were screened after limited dilution and screened by both ELISA and flow cytometry.

RNA  And RT - PCR

Total RNA from various murine tissue samples or purified hematopoietic cell types was obtained using the Trizol (TM) (Invitrogen) method according to the company's instructions. The cDNA was prepared using the iScript TM cDNA synthesis kit (Bio-rad). To amplify the PD-L3 or VISTA total length, the same amount of tissue cDNA (10 ng) was used for the RT-PCR reaction. The PCR product is observed after running through 1% agarose gel.

Cell flow test  ( Flow Cytometry )

Cell flow testing was performed in FACSCAN using CellQuest software (BD Bioscience). Data analysis was performed using FlowJo software (Treesta).

Cell preparation

Total CD4 + T cells were isolated from naive mice using a total CD4 + T cell isolation kit (Miltenyi). When displayed, enriched CD4 + T cells were classified into the naive (CD44low CD25- CD62Lhi) and memory (CD44hi CD25- CD62Llow) populations by flow. In the in vitro proliferation test, CD4 + T cells were labeled with 5 μM CFSE (Molecular Probes) for 10 min at 37 ° C and washed twice before stimulation.

In vitro  Plate-Binding T Cell Activation Assay ( In vitro plate - bound  T cell  activation assay )

Purified CD4 + T cells (100,000 cells per well) were cultured in 96x flat bottom well plates in the presence of anti-CD3 (2C11 clone) and PD-L3 or VISTA-Ig or control-Ig at the indicated ratios. For example, for the full range of titration, 96-well plates were incubated with PD-L3 or VISTA-Ig or control-Ig protein at 1.25 ug / ml (2: 1 ratio), 5 ug / / ml (1: 2 ratio) or 2.5 μg / ml (1: 4 ratio) at 4 ° C overnight with 2.5 μg / ml of αCD3. Wells were washed 3 times with PBS before addition of CD4 + T cells. The replicate cultures were incubated with complete RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM HEPES, 50 μM β-ME, penicillin / streptomycin / L-glutamine Lt; / RTI > When indicated, an appropriate amount of 100 U / ml human IL-2 (PeproTech) or □ CD28 (clone PV-1, BioX cell) was coated with □ CD3 to determine the inhibitory effect of PD-L3 or VISTA- . The medium was analyzed on the third day or the indicated time period in the CFSE profile.

Bone marrow origin DCs , Retroviral transduction, and transduction CD4 + T cell stimulation

Bone marrow-derived DCs have been shown to be highly expressed in large quantities of highly developed methods (Lutz, MB, Kukutsch, N., Ogilivie, AL, Rossner, S., Koch, F., Romani, N., and Schiuler, G. pure dendritic cells from mouse bone marrow. J Immunol Methods 223, 77-92; Son, Y. I., Egawa, S., Tatsumi, T., Redlinger, R. E. Jr., Kalinski, P. and Kanto, T. (2001) A nocel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J Immunol Methods 262, 145-157. Briefly, on day 0, bone marrow cells were separated by flushing from the tibia with a 27G needle from the femur. After red cell dissolution, 1-2 x 106 bone marrow cells were resuspended in 6x well cell culture plates (Nunc, Inc.) in 1 ml complete RPMI 1640 with 20 ng / ml GM-CSF (Prpeotech Inc). A 2 ml supernatant containing either RFP or PD-L3 or VISTA-RFP retrovirus was added to the bone marrow cells. In addition, polybrene (Sigma) was added to a final concentration of 8 μg / ml. Infection was carried out by rotating the plate at 2000 rpm for 45 min at RT. The cells were then incubated for another 2 hours and then fresh medium was added. A similar infection process was repeated on day +1, day +3, day +5, day 7. Loosely adherent cells (90% CD11c +) were obtained on day + 10 and used to stimulate transduced OT-II CD4 + T cells by classifying CD11c + RFP + double positive cells. In the OT-II T cell proliferation assay, 100,000 CFSE-labeled OT-II CD4 + T cells were incubated with 30,000 sorted RFP + or PD-L3 or VISTA-RFP + BMDCs in the presence of the synthesized OVA323-339 peptide (Anaspec) And cultured in 96 well round-bottom plates. Proliferation of OT-II T cells was analyzed at 72 hours by examining the CFSE profile.

For immunization PD - L3  or VISTA Expression

To immunize transgenic mice DO11.10 mice, 300 μg OVA (Sigma) was emulsified in CFA (200 μl) and subcutaneously injected into the side of the mice. Draining lymph nodes and non-draining lymph nodes were obtained at the indicated time points. Single cell suspensions were prepared and analyzed by cell flow analysis for the expression of PD-L3 or VISTA and other surface markers.

PD - L3  or VISTA Inhibitory activity

In vitro, the inhibitory activity of PD-L1 with CD4 + and CD8 + T cell antigen receptor transduced T cells and antigen stimulation was demonstrated using antigen presenting cells overexpressing PD-L1 (Carter, et al. ) Eur J. Immunol. 32: 634-43). Similarly, the lentivirus expressing the PD-L3 or VISTA battlefield disclosed herein was introduced into cell lines expressing major histrocompatibility comlex class II and MHC class I. The response of TEa Tg or the 2C transduced T cells to antigen presented by the empty vector-transduced or PD-L3 or VISTA-transduced antigen presenting cells was determined according to established methods.

Protein expression. Expression patterns in lymphocytes, monocytes and dendritic cell subpopulations as well as non-hematopoietic tissues can be analyzed by RT-PCR and Western blot analysis in combination with alpha PD-L3 or VISTA antibodies disclosed herein using a general protocol Lt; / RTI >

Monoclonal antibody production. PD-L3 or VISTA is overexpressed in mouse B cell line A20, and the recombinant cells are used to immunize Armenian hamsters. After 5x cell immunization, hamsters were augmented with purified PD-L3 or VISTA-Ig fusion proteins that were emulsified in CFA. After 4 weeks, the final enhancement was provided with soluble PD-L3 or VISTA-Ig. Thereafter, fusion of SP2 / 0 and hamster spleen cells was performed on day 4. Sixteen different clones were identified in which PD-L3 or VISTA-Ig fusion protein was recognized by ELISA and stained with PD-L3 or VISTA, but PD-L1 overexpressed in mouse T cell line EL4 was not stained. Eleven of these clones were successfully subcloned and prepared to stain endogenous PD-L3 or VISTA of their cells and assess their ability to block PD-L3 or VISTA function.

Proliferation test

In vitro CD4 + T cell proliferation assay was designed to screen for PD-L3 or VISTA mAb activity. In this test, T cells were stimulated by fixed anti-CD3 in microplate wells, which cross-links with T cell receptors. Activity of PD-L3 or VISTA mAb was detected in two different forms using PD-L3 or VISTA-Ig fusion proteins, including extracellular domains of PD-L3 or VISTA fused with the Fc portion of human IgG . First, when the mAb was co-immobilized with [alpha] CD3, only in the presence of the added soluble PD-L3 or VISTA-Ig fusion protein, this strongly inhibited T cell proliferation. This activity is determined by the ability to bind mAbs immobilized to the wells of PD-L3 or VISTA-Ig. Using this form of the test, clones with high, moderate or low inhibitory activity were identified. Second, when mAb is added as a soluble reagent to the test, it shows strong inhibitory activity by synergy with the immobilized PD-L3 or VISTA-Ig fusion protein in T cell proliferation. In this type of test, clones with various inhibitory activities were identified.

Example  One: PD - L3  or VISTA of Cloning  And sequencing

PD-L3 or VISTA and Treg-sTNF were identified by the global transcription profile of Tregs activated by resting Tregs, αCD3-activated Tregs, αCD3 / αGITRs. ΑGITR was selected for this analysis (Shimizu, et al. (2002), supra) because inducing GITR in Treg appears to destroy their contact-dependent inhibitory activity. PD-L3 or VISTA and Tref-sTNF were identified on an AFFIMETRIX® DNA array based on their particular expression pattern (Table 1). PD-L3 or VISTA exhibits increased expression in the < RTI ID = 0.0 > aCD3 < / RTI > activated Treg and exhibits reduced expression in the presence of? GITR; And Treg-sTNF show an increase in [alpha] CD3 / [alpha] GITR-dependent expression.

For real-time PCR analysis, purified CD4 + CD25 + T cells were stimulated with? CD3, or? CD3 /? GITR, and isolated RNA, overnight, overnight in the medium. The listed expression is relative to actin.

Affrmetrix analysis of the active (vs.) resting CD25 + CD4 + nTreg has sequence homology to the Ig counterpart, but exhibits the expression of an unknown function gene product (RIKEN cDNA 4632428N05 or 4632428N05Rik).

More specifically, the 930 bp gene product was cloned from the CD4 + T cell cDNA library, which is consistent with the predicted size and sequence. Silico-sequence and structural analysis predicted that the protein was 309 amino acid transmembrane proteins with 159 amino acid extracellular domains, 22 membrane amino acid transmembrane domains, and 95 amino acid cytoplasmic tail after maturation (Figure 1 ). The amino acid sequence is homologous to the B7 family ligands such as PD-L1, PD-L2, B7-H3 and B7-H4 as well as the B7 family receptors (ie PD-1, CTLA-4, CD28, BTLA, ICOS) (Ig) -V-like domain having the amino acid sequence of SEQ ID NO: 1 (Fig. 1B-1C). Although the sequence identity of the Ig-V domain between B7-based ligands and receptors is generally not very high (< 40%), the Ig-V domain of 4632428N05Rik binds to the B7-based ligand PD-L1 and PD- . In addition, the sequence shows some highly conserved cysteins (Fig. 1b), important for the formation of intra-chain disulfide bonds (Fig. 1b), which is characteristic of the B7-based ligands (Sica et al. (2003) Immunity 18, 849-861).

The extracellular domain of 46322428N05Rik contained only the Ig-V domain but lacked the Ig-C domain (FIG. 1b-1c). This unique feature is characteristic of B7 family receptors, including both Ig-V and Ig-C domains, which distinguish 4632428N05Rik from all other B7 family ligands (Freeman, G. J (2008) Proc Natl Acad Sci USA 105, 10264-10276; Lazar-Molnar et al., (2008) Proc Natl Acad Sci USA 105, 10483-10488; Lin et al., (2008) Proc Natl Acad Sci USA 105, 3011-3016; Schwartz et al (2001), Nature 410, 604-608; Stamper et al., (2001), Nature 410, 608-61). Consistently, Phylogenetic Maximum Likelihood using the PhyML algorithm positions 4632428N05Rik evolutionarily closer to the B7 family of receptors, particularly PD-L1 than the B7 family ligand (Fig. 2) (Guindon, S , and Gascuel, O. (2003). A simple, fast, and accurate alorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696-704). However, the cytoplasmic tail of PD-L3 or VISTA does not contain the signaling domain (ITIM, ITAM or ITSM, for example) which is the marker domain of B7 family receptors (Sharpe, AH, and Freeman, GJ 2002 ) The B7-CD28 superfamily, Nat Rev Immunol 2, 116-126). Therefore, despite the close evolutionary relationship with the inhibitory receptor PD-1, 4632428N05Rik indicates a novel member of the B7 ligand example. Based on these structural and phylogenetic characteristics, the molecule is named PD-1-expressing ligand (PD-L3 or VISTA). In addition, PD-L3 or VISTA is highly conserved between mouse and human orthologs and shares 77% sequence homology (Fig. 1d).

The nucleic acid sequence encoding mouse PD-L3 or VISTA is set forth in SEQ ID NO: 1 and the mouse PD-L3 or VISTA protein sequence is set forth in SEQ ID NO: 2. The human homologue of PD-L3 or VISTA is located at chromosome 10 (72.9 Mb) and produces a transcript of 4689 bases in length that encodes 311 residue proteins, including 6 nucleoli. The human homolog mRNA coding sequence is provided in the GENBANK accession number NM_022153, and the protein sequence is given in NP_071436. The nucleic acid sequence encoding human PD-L3 or VISTA is herein disclosed in SEQ ID NO: 3 and the human PD-L3 or VISTA protein sequence is set forth in SEQ ID NO: 4. Mouse and human genes share 74% homology and have 68% identity at the protein level. In addition, the homologue is identified on chromosome 20 (27.7 Mb; GENEBANK session number BC098723) or Fugu rubripes and Danio reerio of Rattus norvegicus. In certain embodiments, the PD-L3 or VISTA proteins of the invention share a common amino acid sequence as shown in SEQ ID NO: 5.

Example  2: RT - PCR  Analysis and In cell flow analysis  by PD - L3  or VISTA Expression

As shown in the experiment of FIG. 3, RT-PCR analysis was used to determine the mRNA expression pattern of PD-L3 or VISTA in mouse tissues (FIG. 3A). PD-L3 or VISTA is expressed in tissues (ie, lungs) that are almost infiltrated with hematopoietic tissues (spleen, thymus, bone marrow) or white blood cells. Weak expression is also detected in non-hematopoietic tissues (i.e., heart, kidney, brain, and ovaries). Analysis of some hematopoietic cell types shows expression of PD-L3 or VISTA in peritoneal macrophages, spleen CD11b + monocytes, CD11c + DCs, CD4 + T cells and CD8 + T cells, but low expression in B cells 3b). This expression pattern can be expressed in a gene array database (Gen et al., 2002, Proc Natil Acad Sci USA 99, 4465-4470) of the Genomics Institute of Novartis Research Foundation (GNK) as well as NCBI GEO (Figures 4a-4h).

To study the protein expression, PD-L3 or VISTA specific hamster 8D8 and 6E7 monoclonal antibodies were produced. The specificity was demonstrated by positive staining in PD-L3 or VISTA-overexpressing mouse EL4 T cells, but negative staining in PD-L1-overexpressing EL4 cells (Fig. 5).

Both polyclonal and monoclonal antibodies were made for PD-L3 or VISTA. Using rabbit anti-PD-L3 or VISTA antibodies, PD-L3 or VISTA protein was located in the lymphocyte organs and was significantly found in brain tissue. As the identified monoclonal antibody, the specificity of alpha PD-L3 or VISTA clone D8D was further investigated. In this test, the clone 8D8 has a CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, PD-L3 or VISTA and hIg The panel was inspected for bonding. The results of this test indicate that 8D8 [alpha] PDL-3 is highly specific for PD-L3 or VISTA.

Specifically, PD-L3 or VISTA expression was analyzed by hematopoietic cell flow assay using anti-PD-L3 or VISTA mAb clone 8D8. Foxp3GFP knock-in reporter mice were used to differentiate CD4 + nTreg (34). In peripheral lymphoid organs (spleen and lymph nodes), significant expression was observed in all CD4 + T cell subpopulations (all CD4 + T cells or Foxp3-naive T cells and Foxp3 + nTreg and memory CD4 + T cells) Exhibit significantly lower surface PD-L3 or VISTA expression (Figure 3c). In the thymus, PD-L3 or VISTA expression is negative in CD4 + CD8 + double positive thymocytes, low expression in CD4 monocytic cells, and detectable expression in CD8 monocytic cells. Next, the strong association of high PD-L3 or VISTA expression with CD11b markers is seen in both spleen and peritoneal cells, including F4 / 80 macrophages and myeloid CD11c + Dcs (Fig. 3d-3e). On the other hand, B cells and NK cells are usually negative for PD-L3 or VISTA expression. A small proportion of Gr-1 + granulocytes also express PD-L3 or VISTA (Figure 3f).

Different expression patterns are seen in the same cell lineage from different lymphoid organs (Figure 3g). For CD4 + T cells and CD11b intermediate mononuclear cells, the expression level follows mesenteric lymph node> malignant lymph node and spleen> abdominal cavity and blood. This aspect is less obvious in CD11bhi cells. These data suggest that PD-L3 or VISTA expression in certain cell types can be regulated by cell maturation and / or tissue microenvironment.

In addition to freshly isolated cells, PD-L3 or VISTA expression was also analyzed in spleen CD4 + T cells, CD11bhi monocytes and CD11c + DCs (FIG. 6), without activation and activation in vitro media. Splenocytes were cultured with the medium or incubated with anti-CD3 (for T cell activation) or with IFN? And LPS (for activation of protein spheres and DCs) for 24 hours and then stimulated with PD-L3 VISTA and other B7-based ligands (e.g., PD-L1, PD-L2, B7-H3 and B7-H4). These comparisons showed distinct expression patterns between the molecules. Regardless of activation, expression of PD-L3 or VISTA rapidly disappeared in all cells in vitro media. Conversely, PD-L1 expression was upregulated in CD4 + T cells or CD11bhi monocytes, and CD11c + DCs cultured in medium alone after stimulation, and was further enhanced when stimulated. The expression of PD-L2, B7-H3 and B7-H4 was not significant in the culture conditions used. Deletion of the PD-L3 or VISTA expression in vitro may be specific as compared to other B7-based ligands, but may reflect non-optimized culture conditions that fail to mimic the tissue microenvironment.

To investigate how PD-L3 or VISTA expression is regulated in vivo, CD4 TCR transduced mice DO11.10 were immunized with the conjugated antigenic chicken albumin (OVA) emulsified in complete Freund's adjuvant (CFA) Lt; / RTI &gt; After 24 hours of immunization, cells from draining lymph nodes were examined for PD-L3 or VISTA expression (Fig. 7A). Immunization with only antigen (CFA / OVA) without adjuvant dramatically increased the CD11b + PD-L3 or VISTA + myeloid cell population, which included a mixed population of F4 / 80 macrophages and CD11c + DCs. Furthermore, PD-L3 or VISTA was most up-regulated during the inflammatory immune response (Fig. 7B), even though PD-L1 had the highest, always expressed level of comparison with PD-L1 and PD-L2. In conclusion, this data strongly suggests that PD-L3 or VISTA expression in myeloid APCs is highly regulated by the immune system, and regulates the immune response and contributes to its role in regulating T cell immunity.

In contrast to its increased expression in APCs, PD-L3 or VISTA expression is reduced in DO11.10 CD4 + T cells activated at the later time of immunization (i.e., at 48 hours but not 24 hours). These results suggest that PD-L3 or VISTA expression in CD4 T cells in vivo is regulated by its activated state and the cytokine microenvironment during the active immune response.

Example  3: CD4 + And CD8 + T cell response PD - L3  or VISTA  Functional effects of signal transduction

PD-L3 or VISTA-Ig fusion proteins were produced to investigate the regulatory role of PD-L3 or VISTA in CD4 + T cell responses. The PD-L3 or VISTA-Ig fusion protein comprises the extracellular domain of PD-L3 or VISTA fused to a human IgG1 Fc region. When immobilized on a microplate, PD-L3 or VISTA-Ig, but not the control Ig, as determined by stationary cell division, proliferated macro-purified CD4 + and CD8 + T cells against plate-bound anti-CD3 stimulation (Figs. 9A-9B). The PD-L3 or VISTA-Ig fusion protein does not affect the absorption of the anti-CD3 antibody into the plastic well as determined by ELISA (data not shown), thus eliminating the possibility of nonspecific inhibitory effects . PD-1 KO CD4 + T cells were also inhibited (Fig. 9c), indicating that PD-1 is not a receptor for PD-L3 or VISTA. The inhibitory effects of PD-L1-Ig and PD-L3 or VISTA-Ig were also directly compared (FIG. 10). In order to stimulate CD4 + T cells, PD-L3 or VISTA-Ig shows a similar inhibitory effect to PD-L1-Ig fusion protein when an appropriate amount of Ig fusion protein together with CD3 is absorbed into the microplate.

The effects of PD-L3 or VISTA-Ig in the subtypes of sorted naïve (CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) CD4 + T cells were investigated because large purified CD4 + T cells have diverse subgroups 11). This is less effective than in memory cells, but shows that PD-L3 or VISTA can inhibit the proliferation of both subpopulations.

In order to better understand the mechanism of PD-L3 or VISTA-mediated inhibition, the expression of early TCR activation and apoptosis markers was measured after activation of T cells in the presence or absence of PD-L3 or VISTA-Ig. There was global inhibition in the expression of early activation markers, CD69, CD44 and CD62L (supplemental 12a), consistent with the negative effect on cell proliferation. On the other hand, PD-L3 or VISTA-Ig fusion protein did not induce apoptosis. Conversely, in both the early (24 h) and post (48 h) TCR activation, less apoptosis (by percentage of annexin V + 7 AAD-cells) in the presence of PD-L3 or VISTA or VISTA- (Fig. 12B). For example, in the 24 hour period, ~ 27% cells in total "ungated" group were apoptotic in the presence of PD-L3 or VISTA or VISTA-Ig, whereas ~ 39% of control cells were apoptotic . Approximately 72.6% of the control cells were apoptotic cells when treated with PD-L3 or VISTA and VISTA-Ig, whereas only 43.5% cells were PD-L3 It is clear that VISTA or VISTA-Ig strongly inhibited activation-induced-cell death (ACID). Similar results were obtained at 48 hours. Thus, it has been shown that PD-L3 or VISTA or VISTA suppresses early TCR activation and arrests cell division, but regulates CD4 + T cell responses spontaneously with minimal direct effect on apoptosis. The mechanism of inhibition is similar to that of B7-H4 (Sica, GL, Choi, IH, Zhu, G., Tamada, K., Wang, SD, Tamura, H., Chapoval, AI, J., and Chen, L. (2003) B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 18, 849-861).

Two-step testing has been developed to determine whether PD-L3 or VISTA or VISTA-Ig can inhibit pre-activated CD4 + T cells, and how its inhibitory effect persists. The inhibitory effect of PD-L3 or VISTA or VISTA-Ig fusion protein was shown to persist until after its removal 24 hours after activation (Fig. 9d). In addition, both naive and pre-activated CD4 + T cells can be inhibited by PD-L3 or VISTA or VISTA-Ig (i in Figure 9d, iii in Figure 9d and iv in Figure 9d).

Next, the effect of PD-L3 or VISTA or VISTA-Ig on the production of cytokines of CD4 + T cells was analyzed. PD-L3 or VISTA or VISTA-Ig inhibited the production of Th1 cytokines IL-2 and IFN alpha from cultures of macro-purified CD4 + T cells (FIGS. 13A-13B). The effects of PD-L3 or VISTA or VISTA-Ig were further examined in a separate population of naïve (CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) CD4 + T cells. Membrane CD4 + T cells are a major source for the production of cytokines within the CD4 + T cell portion, and PD-L3 or VISTA or VISTA can inhibit this production (Figures 13C-13D). Similar inhibitory effects of PD-L3 or VISTA or VISTA were also shown in the production of IFN alpha from CD8 + T cells (Figure 13E). The inhibitory effect of PD-L3 or VISTA or VISTA on cytokine production by CD4 + and CD8 + T cells is consistent with the hypothesis that PD-L3 or VISTA or VISTA downregulates the immune response.

Next, a study was designed to determine the factors that could overcome the inhibitory effects of PD-L3 or VISTA or VISTA. When PD-L3 or VISTA or VISTA inhibits IL-2 production and IL-2 is thought to be important for T cell survival and proliferation, IL-2 may bypass the inhibitory activity of PD-L3 or VISTA or VISTA I can build a family. Unlike IL-15, IL-7 or IL-23, exogenous IL-2 reverses the inhibitory effects of PD-L3 or VISTA or VISTA-Ig partially in cell proliferation, as shown in FIG. 14A. Incomplete overcome by high levels of IL-2, signaling of PD-L3 or VISTA simply targets a wider T cell activation pathway than IL-2 production. On the other hand, the strong co-stimulatory signal provided by the anti-CD28 agonistic antibody completely reverses the PD-L3 or VISTA or VISTA-Ig mediated inhibition (FIG. 14b), whereas the median level of co- stimulation is PD- Or VISTA signaling (Fig. 14C). These results suggest that PD-L3 or VISTA or VISTA-mediated immunosuppression may be more effective under low inflammatory disease, but will inevitably be overwhelmed by strong positive co-stimulation. Here, PD-L3 or VISTA or VISTA share this characteristic with other inhibitory B7-based ligands such as PD-L1 and B7-H4 (Sica et al., (2003) Immunity 18, 849-861 .; Carter et al., (2002), Eur J Immunol 32, 634-643).

PD-L3 or VISTA or VISTA or VISTA during the interactions between APCs and T cells expressed in APCs as well as PD-L3 or VISTA or VISTA-Ig fusion proteins can inhibit antigen-specific T cell activation It is necessary to confirm. For this purpose, PD-L3 or VISTA or VISTA-RFP or RFP control proteins were overexpressed through retroviral transduction into an artificial antigen presenting cell line (CHO-APC) stably expressing MHCII and B7-2 molecules (Latchman et al., (2001), Natu Immunol 2, 261-268). One problem with PD-L3 or VISTA or VISTA expression in CHO is that the majority of PD-L3 or VISTA or VISTA fail to locate on the cell surface, presumably because of PD-L3 or VISTA or VISTA surface localization (The data is not shown). Although there is no conspicuous motif present in the cytoplasmic tail of PD-L3 or VISTA or VISTA, which may represent a regulatory action, it is believed that the tail plays an important role in its cellular localization. As a result, a tail-free PD-L3 or VISTA or VISTA mutation was devised and found to successfully localize on the CHO cell surface.

To stimulate T cell responses, CHO-PD-L3 or VISTA or VISTA or CHO-RFP cells were incubated with DO11.10 CD4 + T cells in the presence of antigenic OVA peptide. As shown in FIGS. 15A-15C, CHO-PD-L3 or VISTA or VISTA induced a lower DO11.10 proliferation than CHO-RFP cells. Consistent with the fact that stronger stimulatory signals could overcome the inhibitory effects of PD-L3 or VISTA or VISTA, this inhibitory effect was more pronounced at lower peptide concentrations.

In addition, inhibitory effects of PD-L3 or VISTA or VISTA full-length in native APCs were confirmed. Bone marrow-derived dendritic cells (BMDC) cultured in vitro do not express high levels of PD-L3 or VISTA or VISTA (FIG. 16). During the 10 day incubation period, PD-L3 or VISTA or VISTA-RFP or RFP was expressed in BMDCs by retroviral transduction. The transduced cells were classified for homogenity based on RFP expression. Expression levels of PD-L3 or VISTA or VISTA in transduced DCs were measured by staining with anti-PD-L3 or VISTA or VISTA mAbs and the levels in freshly isolated peritoneal macrophages within physiological expression range (Fig. 16). The sorted BMDCs were then used to stimulate OVA-specific transduced CD4 + T cells (OTII) in the presence of OVA peptide (Figure 15d). There, PD-L3 or VISTA or VISTA expression in BMDCs was shown to inhibit the proliferative CD4 + T cell proliferative response. The results are consistent with previous data using PD-L3 or VISTA or VISTA-Ig fusion proteins and CHO-APC cells indicating that PD-L3 or VISTA or VISTA can inhibit T cell-mediated immune responses present.

Example  4: Multiple sclerosis animal model ( EAE ) &Lt; / RTI &gt; PD - L3  or VISTA  Or VISTA antibody

Since PD-L3 or VISTA or VISTA mAbs appear to inhibit T cell responses in vivo, PD-L3 or VISTA or VISTA were tested to assess whether this could inhibit T cell-mediated autoimmunity. Experimental allergic encephalomyelitis (EAE) model was used to determine the functional effect of PDL-L3 mAbs in inflammatory diseases. EAE can be induced by immunization with myelin antigen in the adjuvant or adoptive transfer of myelin-specific T cells, which can be induced by various functional T cells and B cells, and inflammatory infiltration of macrophages And demyelination of the central nervous system.

To avoid anaphylaxis caused by the injection of large amounts of mAbs as external antigens, the [alpha] PDL-L3 mAb was tested in the passive EAE model. In this adoptive transfer EAE model, donor SJL mice were immunized with CFA and PLP peptides. On day 10, total lymphocytes from multiple lymph nodes were isolated and cultured for 4 days in vitro with PLP peptide, IL-23 (20 ng / ml) and anti-IFNg (10 μg / ml). The expanded CD4 + T cells were then purified and transferred to adoptive recipients in naive recipient mice. This analysis implies that the [alpha] PDL-L3 mAb not only delayed the onset of the disease, but also significantly shifted the course of the disease by reducing the severity of the disease (Fig. 17). It also reduced the severity at high rates of the mice and significantly increased the survival rate from about 22% to 75% or more. The activity of the alpha PDL-L3 mAb in EAE indicates that it is consistent with the above in vitro data and indicates the use of the reagent as a novel inflammation modulating agent in a variety of inflammatory diseases.

Example  5: CNS In VISTA Manifestation of

The expression of VISTA in the CNS was also effective. This test shows that VISTA expression significantly reduced CD11b + cells in mice with disease (76% 33%) (FIG. 23), consistent with the hypothesis that deletion of VISTA would result in increased inflammation. This is interesting and is functionally significant when compared with inflammatory myeloid cells with MDSC in tumors expressing VISTA at a very high level. It has been reported that EAE mice have an increased number of myeloid derived suppressor cells (CD11b + Ly-6Chigh MDSC), which strongly suppresses T cell activation, in the spleen and can alleviate disease 32. The data strongly support that VISTA can function in myeloid-mediated suppression in EAE.

Example  6: EAE On the fate and function of T cells in VISTA Influence of

In addition, an experiment was conducted to examine the effect of VISTA on the fate and function of T cells in EAE. We determined whether VISTA changes pathogenicity, development of CSF T cells, expansion of clonal T cells, polarity of T cells, longevity and conversion of Teff ⇒ Treg. The blocking effect of VISTA on T cell fate in EAE was studied. Consistent with higher disease scores, CNS analysis at the end of the disease course identified significantly more IL-17A-producing CD4 + T cell infiltration (0.66 ⇒ 11%) in the 13F3 (VISTA) treated group (Figure 24) .

Example  7: PD - L3  or VISTA  or VISTA  Transduction ( transgenic ) And knock-out mice

Using the lentivirus infection of embryos, four transgenic mice were produced that always express PD-L3 or VISTA or VISTA. The mice express the full length of PD-L3 or VISTA or VISTA under the control of the human elongation factor 1 promoter. The mice were produced using the lentiviral pWPT vector. PD-L3 or VISTA or VISTA co-stimulate αCD3 T cell proliferation in vitro, similar to other members of the PD-L1 system (Appay et al. (2002) J. Immunol. 168: 5954-8) , But is considered to act as a negative regulator in vivo. In this regard, the mice are expected to spontaneously develop autoimmunity, and the in vivo immune response (i.e. humoral immune response, T cell priming) in PD-L3 or VISTA or VISTA transgenic mice is systemic It was investigated to evaluate autoimmune disease development.

For knockout mice, they are inactivated spontaneously by autoimmunity and homologous recombination. BAC clones containing PD-L3 or VISTA or VISTA full-length sequences were purchased from INVITROGENTM (Carlsbad, Calif.). The PD-L3 or VISTA or VISTA target vector is a vector containing a 1.6 kb fragment located on the 5 'side of PD-L3 or the second exon of the VISTA or VISTA gene on a neomycin gene and a third exon of PD-L3 or VISTA or VISTA 3 ' side of the neomycin gene. B6-derived embryonic stem (ES) cells were electron invaded using PD-L3 or VISTA or VISTA target vectors and recombinant clones were screened. The selected clones were then injected into C5BL / 6 undifferentiated cells (blastocyte) and the resulting chimeric male offspring was matched with FLP-deleter mice to remove the neomycin cassette. Delivery of the targeted allele of the offspring was determined by PCR from the genomic DNA. The second and third exons include the PD-L3 or VISTA or VISTA domains, and thus the resulting mice have only the inactivated form of PD-L3 or VISTA or VISTA molecules.

The overall immune capacity of PD-L3 or VISTA or VISTA deficient mice is determined by measuring the T cell response to the antigen, humoral immune response, external autoimmunity (e.g., systemic lupus erythematosus, inflammatory bowel disease) / RTI &gt; - / - mice, including increased susceptibility to other autoimmune diseases (experimental autoimmune encephalomyelitis) (Chen (2004)).

Example  8: Collagen-induced arthritis examined in animal models PD - L3  or VISTA  Or VISTA specific antibody

As shown in the experiment in Figure 18, male DBA / 1J mice were injected with 100 μl of the emulsion containing 100 μg chicken type-II collagen (C-II) (Mycobacterium tuberculosis 3.5 mg / ml) And IP-fortified on day 21 after immunization with 100 [mu] g water-soluble C-II. The mice in each treatment group (n = 6) were either untreated (NT-black circles), 300 μg (red triangles) of monoclonal antibody "7c9" or 300 μg hamster IgG (Ham Ig- EH was treated as one of "13F3" (green triangle). Injection was given every 2 days. The swelling of the joints was scored on a scale of 0-4 for each foot of each mouse on the day indicated. The arthritis score shown above is the score of all feet in each treated group of mice divided by the number of mice in the group.

Example  9: VISTA  By monoclonal antibody VISTA  Blocking From the examiner  T cell response.

The VISTA-specific mAb (13F3) was identified as neutralizing VISTA-mediated inhibition (Figure 19). To stimulate OT-II transduced CD4 + T cells in the presence or absence of 13F3, CD11bhi myeloid APCs were purified from naïve mice. Consistent with its neutralizing effect, 13F3 promoted T cell proliferation stimulated by CD11bhi myeloid cells, which were shown to be expressed at high levels of VISTA.

Example  10: anti- VISTA Enhances anti-tumor immunity.

Because of its ability to enhance T cell activation of anti-VSTA, we investigated whether anti-VSTA enhances protective immune response against immunogenic tumors. A good experience model is bladder carcinoma, MB49. MB49 expresses male antigens, so if the intermediate immunogenicity is in the female and there is no immunological intervention, it grows and kills the female. To test the efficacy of VISTA treatment, MB49 tumor cells were injected subcutaneously (sq) into female mice and treated with □ VISTA. The size of the tumor was determined daily until the mice had to be euthanized. As can be readily seen in FIG. 20, anti-VSTA therapy greatly impairs tumor growth. Which is believed to be due to its ability to enhance the cell-mediated immunity (CMI) response of anti-VSTA.

Example  Tumor Mitigation in a 11: 4 Mouse Tumor Model tumor regression ) On the effect of VISTA

In experiments with immunogenic bladder cancer MB49, we have shown that neutralization of VISTA with mAb 13F3 protects the host from tumor growth. The data indicate that VISTA plays a significant role in the regulation of negative immunity due to its very high expression in MDSC in the microenvironment of the tumor. Studies investigating the effects of anti-mouse VISTA in immunogenicity (MB49) and very non-immunogenic (B16) tumor models further suggest that the effects of αVISTA treatment may explain the mechanism of action and provide the basis for optimal dose and time zone selection Respectively. The basis for each tumor model is as follows.

MB49 in female mice has already been shown to be effective in these mouse models. In this model, MDSC also expresses elevated levels of VISTA (data not shown). In this model, because of the presence of the H-Y antigen, the MB49 tumor has intermediate immunogenicity. Since anti-VSTA treatment was found to be effective, the dose of anti-VSTA treatment (1-100 μg / mouse); And the time of day (day of tumor administration, or the 4th, 7th, and 10th day of tumor, therapeutic intervention)] will be used as a "positive" control.

Effective doses and time zones in MB49: female mice in male mice were used to determine the effectiveness of anti-VSTA (tumor less immune) in male mice.

The B16 melanoma: anti-CTLA-4 mAb appeared to be highly effective in this model, and the mouse model showed a non-immunogenic tumor where it is valuable to predict success in humans. Capacity usage and time zones will be similar to those seen in the MB49 model.

ID8 Ovarian cancer: In this model, VISTA expression was dramatically shown to be higher in MDSCs. Mice bearing ID8 tumors were treated with αVISTA at the time of tumor injection, or at days 5, 15, and 25 after injection.

Way. B6 WT mice were used to determine the optimal dose and time zone of anti-VSTA treatment for alleviation of all the mouse tumor models described above. The models used are listed in the table above.

The readout for this dose and time of day testing is tumor growth kinetics. For the MB49 and B16 studies, all tumor studies were performed via intradermal (i.d.) infusion and thus tumor size can be easily measured. Tumor measurements were collected every 2-3 days using a caliper. In each of the above models, the effect of anti-VSTA or control antibody will be examined for its ability to slow down tumor growth or to promote tumor alleviation. The growth of ID8 will be followed using luciferase transduced ID8 and the entire body will be imaged using the IVIS Workstation. The survival of the host will also be determined.

Data on tumor growth are expressed as mean tumor volume ± SEM, and differences between groups will be analyzed by a two-tailed AVONA (two-tailed ANOVA). The probability (p) values below 0.05 are statistically significant. Survival data were analyzed using Kaplan-Meier with the Wilcoxon rank and log-rank used to confirm significance of survival between groups. In the B16 model, the frequency of mice developing vitiligo is determined.

Using this method, delayed tumor growth and / or tumor relief in mice was obtained in comparison to mice treated with control Ab in some non-immunogenic tumor models. It has already been shown that anti-VSTA treatment delays tumor growth in immunogenic tumor models. Because each of these tumor models has their own specific growth kinetics and predicts their dependence on VISTA to provide tumor growth and inhibit immunity, it is possible that mAb Lt; / RTI &gt; In addition, at least three different concentrations of the VISTA mAb will be examined to determine the optimal concentration for therapeutic benefit.

As shown in FIGS. 21A-21E, VISTA mAb treatment reduces tumor growth in the four tumor models, where mice a. MB49, b. MCA105, or c. Subcutaneously (sq) into EG7 tumor cells, or d. ID-8-luciferase tumor cells were injected intraperitoneally (ip) and treated with VISTA mAb 13F3 every other day (300 μg) starting on day +1. Subcutaneous tumor growth was monitored. For ID8-luciferase tumors, mice were imaged on day 30 using Xenogen IVIS. e. Expression of VISTA in the myeloid leukocytes of mice bearing tumors was also determined. Multiple lymph nodes (LN) and tumor tissue (ascites) were analyzed for VISTA expression. This finding indicates that VISTA expressed in MDSCs, which interfere with the development of protective anti-tumor immunity, is a major inhibitory molecule, and that VISTA relaxes this inhibitory activity, permits immune mediation and slows tumor growth. In addition, the findings support the conclusion that VISTA in myeloid cells is an essential function in regulating the degree of inflammation in autoimmune diseases.

Example  12: Treatment of autoimmune diseases Oligomer VISTA  And VISTA  Synthesis of fusion proteins

In vitro soluble VISTA-Ig is not inhibitory and can not bind to easily detected cells. In contrast, the molecules bound to plastics are very inhibitory. In addition, studies using in vivo VISTA-Ig show no apparent activity (data not shown). For this study, the created VISTA-Ig has a mutation in the CH2-CH3 domain that interferes with FcR binding and is therefore not cytoplasmically compatible in vivo. Recent studies have shown that binding of PD-1 and quadruple PD-L1 is 100 times higher than that of monomer PD-L126 (Kd 6x10-8 M) and binding to cells is easily detectable. 4-coupled PD-L1 can be tested in vivo but appears to block functional inhibition by the native PD-L1 in vitro. Using a similar method, oligomers are created that target the VISTA pathway and induce potent immunosuppressive activity in vitro and in vivo.

The oligomer was prepared using a monomeric extracellular domain of VISTA or a fragment thereof, e.g., at least 50, 75, 100, 125, 150, 175 or 200 amino acids in length, and the extracellular domain, or portion thereof, As a building block. In this way, the present invention has the advantages of a well-established MHC 4 coalescence technique. In this way, the VISTA ectodomain structure or fragment can be used to generate a series of VISTA complexes with a valency of 4 to 7 valency, wherein the N-terminal of the various domain oligomerization (identified above) Lt; / RTI &gt;

Thus, a series of non-covalent oligomers can be made based on a very high affinity or coiled-coil domain that aids in the stable formation of bidentate, tridentate, quaternary, quaternary, and hexahedral assemblies. The oligomeric structure is expressed in host cells, such as E. coli. When the expression is effective in E. coli, the expressed oligomer is refolded and purified from the inclusion body using a common lab chamber protocol. This approach has produced generally high quality materials for both biological and structural analysis, including MHC-peptide complexes and quadruple GITL66. The separated oligomer proteins were then examined by SDS-PAGE, analytical gel filtration, analytical ultracentrifugation and mass spectrometry. The quality control measurements ensure the availability of homologous well-characterized materials for in vitro and in vitro studies. The parallel structure of the construct results in the same number of molecules as the oligomeric state, since each VISTA complex will productively interact with the VISTA receptor bound to the cell surface. The structure is very stable and uniform in the oligomeric state. (Unshared or twisted coil oligomerization shows melting points in excess of 100 캜, except for the 7-mer sequence in which the molten gum exhibits 95 캜.

VISTA-Ig as well as cytoplasmic or cytoplasmic non-covalent complexes. The Fc fusion constructs of the inframe VISTA with IgG1 Fc (both wild-type IgG1 and the existing non-Fc-bound IgG1) were modified at the N-terminal BirA site for enzymatic biotinylation and the pIRES2-EGFP vector Lt; / RTI &gt; Enzymatic biotinylation allows for specific, single residue modifications and orientation towards the polyaddition of avidin. This approach has been used to produce a variety of Ig-fusion proteins including B7-1, PD-L1, PD-L2 and TIM-3. The expressed proteins were then enzymatically biotinylated in vitro and purified by size exclusion HPLC and quaternized using PE-avidin. The cytosolic or non-cytoplasmic 4-mer of the result is measured in vivo.

Such engineered multimer VISTA proteins are useful for treating autoimmunity and other diseases, wherein mediation and immunosuppression in the VISTA pathway is therapeutically ensured.

Example  13: to induce immunosuppression VISTA Adenovirus  Vector (adenoviral vector )

Gene transplantation using recombinant adeno-associated virus (AAV) has shown great technological advances in gene therapy. AAV-mediated gene delivery of the PD-L1 gene, or CTLA4-Ig and CD30-Ig, has achieved therapeutic efficacy in autoimmune disease models of lupus and heart transplant. The method is used to deliver either the full length of VISTA, or one of the oligomers of the VISTA Eta domain, and their therapeutic effect is measured in the EAE model. A recombinant adenoviral vector expressing either the full length of VISTA or one of the oligomers of the VISTA ectodomain was constructed using the Adeno-XTM expression system (Clontech) according to the manufacturer's instructions. Briefly, VISTA was cloned into the E1 and E3-deleted, pAdDEST-based expression vectors under the human cytomegalovirus (CMV) promoter. Thereafter, VISTA and control lacZ expressing adenovirus were purified from cell lysates. Adenovirus was administered to mice by intratracheal infusion for systemic overexpression of VISTA either before or after the induction of the disease via immunization, or after the onset of the disease [1x109 plaque-typhoid unit (pfu)]. The control mice received 100 [mu] l PBS. Disease development and changes were monitored in SJL mice and C57BL / 6 mice, which exhibit different disease progression patterns and represent two different forms of clinical manifestations in human MS patients.

Example  14: Engineered protein and Adenovirus  Functional study using vector

Mice were also administered with engineered VISTA and / or adenoviral vectors (5-100 μg protein / mouse x 3 weeks). After administration, T cell expansion and differentiation as well as EAE development were determined.

Example  15: VISTA Structural studies of VISTA  Determination of molecular determinants of function

The affinity, specificity, oligomer state and formation and location of the structured signal complex make an important contribution to immune function. Because the structure of the receptor-ligand ectodomain directly regulates the migration, organization and function of noncovalently bound cytosolic signals and scaffolding molecules, all of the above features have an effect on signal transduction and immunomodulation . VISTA's high resolution crystal structure as well as its high-throughput crystalization and structure determination approaches are determined using techniques including bacterial, insect and mammalian expression systems. To validate the graphically observed disulfide bond pattern, a high resolution mass spectrometer was applied using an approach that successfully supported the published studies of TIM-3 and human DcR359. Based on the structural results, mutations were devised near the site of a mutated series with altered oligomeric properties and a perturbed region of the VISTA IgV domain. The mutant proteins will provide additional direct mechanism insight into VISTA function and will be useful in therapy where immunosuppression is desirable where such autoimmune, allergic and inflammatory diseases are identified herein. In order to evaluate the disease outcome, immunosuppressive effects in disease mitigation, such mutations, in particular oligomers, have been examined in vitro and have been used in animal models of autoimmune and inflammatory disease, or in the development of such autoimmune or inflammatory diseases, As well as to protect them.

The oligomeric VISTA protein activates VISTA pathway and function as a target of immune mediation in autoimmunity. Such intervention may have therapeutic effects in autoimmune diseases or other diseases where it is desirable to inhibit immunity and inhibit autoimmunity. This is accomplished by administering oligomerized VISTA proteins in different autoimmune and inflammatory models, such as EAE and collagen-induced arthritis animal models. Also, as described above, adenoviral vectors that overexpress VISTA full-length or oligomers of VISTA are made and tested in vivo. These studies will confirm the immunosuppressive effects of VISTA oligomers.

Example  16: conditional VISTA  Over-expression transduction Mouse note  ( mouse strain ) Was used for the experiment VISTA  Transduction Mouse note : R26StopFLVISTA  ( VISTA ))

The targeting construct containing the full-length cDNA of VISTA, preceded by the loxp-side STOP cassette, was always targeted to the expressing ROS26 position. A number of correctly targeted R26StopFL / -VISTA litters were born and bred at the CMV-Cre remover week 60. GFP and stressed VISTA expression were confirmed by line data on VISTA x CMV-cre. Studies of the immune status of the mice will identify inhibitory phenotypes (T cell responses to antigens, antibody titer, etc.). To determine if the location of VISTA expression affects inhibition, the VISTA line will be crossed with CD4-cre, CD11c-cre and Lys-cre. In addition, the phenotype and function of T cells will be determined, and it will be determined whether overexpression of VISTA will result in the production of aTreg. In this study, in order to see if antigen immunization induces antigen-specific Tregs in the presence of over-expressed VISTA, the OVA-immunocomplexx VISTA strain is adoptively transferred to the WT host. This confirms that VISTA affects Treg differentiation.

In addition, the study was efficacious in the EAE model, and the impact of VISTA protein in different strains on disease development will be assessed (by crossing with CD4-, CD11c-, and Lys-cre). Assuming that the disease can be inhibited by systemic VISTA overexpression or in a CMV x VISTA mutation, the transient modulation of disease development uses Cre-ERT 2x VISTA. Overexpression of VISTA can be induced to prevent overexpression of VISTA through the administration of tamoxifen, to determine whether VISTA affects the induction and / or action steps of the immune system, prior to the onset of the disease, at the onset, or at the disease peak have. Using BM chimeric mice, transiently limited VISTA overexpression may be limited in hematopoietic populations. For an understanding of regulating the time window over which VISTA is overexpressed, VISTA generally occurs and then serologically turns off with administration of the anti-VSTA mAb. The study will determine where and when VISTA has a role in regulating the course and progression of autoimmune diseases.

Example  17: anti- VISTA  Antibody CD40 / TLR  Effect of agonist vaccine

As shown in Figure 22, an experiment was conducted to test the effect of anti-VSTA antibodies on the efficacy of the vaccine. The results showed that anti-VSTA enhances the therapeutic effect of the CD40 / TLR vaccine. C57BL / 6 mice stimulated 1x105 metastatic B16.F10 melanoma cells with sq. Four days later, mice were vaccinated with 100 μg tumor-associated antigen ΔV, 100 μg αCD40 FGK45 (CD40 agonist antibody) and 100 μg S-27609 (TLR7 agonist) with or without anti-VISTA (200 μg x 3 / week) . Tumor growth was monitored by caliper measurements.

As set forth in the present invention, are provided in the following claims. These claims are intended to cover all such general and specific features described herein, as well as all statements of the foregoing, which may be in between, as a matter of language.

                                SEQUENCE LISTING <110> Trustees of Dartmouth College       Noelle, Randolph J.       Lu, Li-Fan       Sergio, Quezada       David, Gondek <120> REGULATORY T CELL MEDIATOR PROTEINS AND USES THEREOF <130> 76799.000106 <140> PCT / US2011 / 030087 <141> 2011-03-25 <150> 60 / 674,567 <151> 2005-04-25 <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 4795 <212> DNA <213> Mus musculus <400> 1 gagcattcac tctagcgagc gagcggcgtg tacagccggc tccctgggct cctggagtcc 60 cgcttgctcc aagcgcactc cagcagtctc tttctgctct tgcccggctc gacggcgaca 120 tgggtgtccc cgcggtccca gaggccagca gcccgcgctg gggaaccctg ctccttgcta 180 ttttcctggc tgcatccaga ggtctggtag cagccttcaa ggtcaccact ccatattctc 240 tctatgtgtg tcccgaggga cagaatgcca ccctcacctg caggattctg ggccccgtgt 300 ccaaagggca cgatgtgacc atctacaaga cgtggtacct cagctcacga ggcgaggtcc 360 agatgtgcaa agaacaccgg cccatacgca acttcacatt gcagcacctt cagcaccacg 420 gaagccacct gaaagccaac gccagccatg accagcccca gaagcatggg ctagagctag 480 cttctgacca ccacggtaac ttctctatca ccctgcgcaa tgtgacccca agggacagcg 540 gcctctactg ctgtctagtg atagaattaa aaaaccacca cccagaacaa cggttctacg 600 ggtccatgga gctacaggta caggcaggca aaggctcggg gtccacatgc atggcgtcta 660          atgagcagga cagtgacagc atcacggctg cggccctggc caccggcgcc tgcatcgtgg 720 gaatcctctg cctccccctt atcctgctgc tggtctataa gcagagacag gtggcctctc 780 accgccgtgc ccaggagttg gtgaggatgg acagcagcaa cacccaagga atcgaaaacc 840 caggcttcga gaccactcca cccttccagg ggatgcctga ggccaagacc aggccgccac 900 tgtcctatgt ggcccagcgg caaccttcgg agtcaggacg gtacctgctc tctgacccca 960 gcacacctct gtcgcctcca ggccctgggg acgtcttttt cccatcccta gatccagtcc 1020 ctgactcccc taactctgaa gccatctaaa ccagctgggg aaccatgaac catggtacct 1080 gggtcaggga tatgtgcact tgatctatgg ctggcccttg gacagtcttt taggcactga 1140 ctccagcttc cttgctcctg ctctgagcct agactctgct tttacaagat gcacagaccc 1200 tcccctatct ctttcagacg ctacttgggg ggcagggaga agatgttgga ttgctcatgg 1260 ctgttctcaa gatcttggga tgctgagttc tccctagaga cttgacttcg acagccacag 1320 atgtcagatg acctgcatcc tatgaacgtc cggcttggca agagcctttc ttcatggaaa 1380 ccagtagccc ggaggggatg aggtaggcac cttgccaccc tcccgggaga gagacacaag 1440 atgtgagaga ctcctgctg ctgtgggggt gtggctggcc tgcttgtttg cctgaggatg 1500 ctcctctgtt ggactgactc tatccccctg gattctggag cttggctggc ctatgtccca 1560 ccagaggagc atctcagcag ccttccacca gcaacctgag ggcctgccag cttcgtggct 1620 ctgggctctc attacctgta tggccgtcca cagagctcag tggccagagg ctttgaaaca 1680 ggaagtacat gtcaggttca ggaaccactg tgagctcatt agtgtcttga gcaatgtgag 1740 gcctggacca gtggacacgg agggagggtg gcgagaggat gatggggatg atgaggggaa 1800 cacgctccct tcctgtcctt gtcatccacc actaccacta ttcagtgtgg agcagtggca 1860 aaggtgaccg acctccacaa tgtcctagtg atgctggacc atttctaagt gtgaaagaga 1920 tgctattaaa aacagtatgt ggcaatggct gccaacagct gagtggactg gaggcactgg 1980 ctttaaggcc ctggaggtgc agggcccggt atggggatag ggatgggagt ttcagtgagg 2040 gcctagggat cactccgctt ctgaccactc ttcttctgag cctcacctca gggtgacctt 2100 caggcacaca gaagagcttg cccctggtcc gatactactc ttggctctca tctccagggt 2160 ttggcatgac ctgggcacac agggggagtc ttcagaaagg attttaaagc atgaaaagaa 2220 agggtagttc ttgtgaggta gggatgggca gctgatgttt gagagtgagg agggatacgg 2280 ctgggcagat cactctccag tctctagagg gaaagtagct ctaagtctgg gagagcagca 2340 gcccagtggt accatatgtc ttcttgcagc ttccactggc tgggctgaac tgggcatggg 2400 taggaaagct cctgttctgg gcctgcagcc agggagaacc ccattcattc cctgaggaca 2460 gatgggtggg gagagaagag agagtttcag gccgggaagc agcaataagc tatctgctgg 2520 ggacccagac aagttgtctg atgaggtcca agatgtggga tgccagttat acctggggct 2580 tggggatcct tagaggcttt gtatcatcat cataggagtg tcggggtggc cagggcatca 2640 aagccatgac ccctgtttta tcctcagggt ccactcttct gcaccatcca ttgctctaga 2700 tctatgcagt tactatagac agaatgtgtt gttctgtttg gctttgggga taatggcctg 2760 gcgaactgcc agctgttcag tggcagggct gtgaggccag tcaaagacta gaacccacag 2820 accagctgaa cgatgagtat agcctgtccc ctgggggagc ctgacctgtc tccagcccta 2880 agcttcagac ctcaccactc agatgacttc taagaatttg cctgtgggga cccctgcatg 2940 gctgcagctc cgtggaaagg agaggaggcc cccagcagaa gaaccactcg cttcctgccc 3000 agcttcctcc tgtagggctc taagtctctt cttcttggga ccctgcaagc aaaggcatgt 3060 cagcttggtg gtttcctgtt ttgggtgaag ttttgtgtgg tccgggttct gtctacatcc 3120 atgaacttgg ggtgctacca ccttgctgct gctgtagaga cagctgcagg atcttagggt 3180 ggaaaatgga ggtgccctga ggtgctagcc cttggggcaa aagatggggt ggcaatgaga 3240 cacttgggg aactgagttc cccaagagga gggaggagcc ctgtagcctc aagggccata 3300 ttgggttcct ggtaccagca aaagcctaga gagcgaagtc tgtattttga ggaggtaatt 3360 gatccttacg gaatccatca gaaatttgga gcgggtgctt tatctatctc tggagggtct 3420 ctacctatct ccgatgaagc tctccctggg cctgggatgg gagaaaccag gaggaaaggt 3480 gtctgataaa gcaggggctt cttgacaagc caaagggcca ctggtagctg ttgtggaccg 3540 agctgaccct gctgaagtat tgtagtgtgc cttggaccaa cttctcaaaa gagcaacccc 3600 ggggctaccc tacttctgcc aggaagaggc ggagaagggg ctgagaggcc tggaaggggc 3660 tagctccttc tttgagaact gctccccgga ggacttggag gaggcggcta ggctacgggc 3720 tgctgagggc cctttgtctt tcctaacctg ggcactgtta ggatgctccc tcctggaaaa 3780 ggctttcctg ggtgtgagct agagcagtgt ccatgccagc gctgaacctg ccatggtggg 3840 agctgaacta aaaatttctc agggaactaa aataggcaaa agaggaactg ggggaggagg 3900 gtgccaggca ggatgggggg aagggagggc agtgcaaaag tctcttgaaa cacagacagc 3960 ccagctgagt gccagtccca gatcacagag aatacggctc atctggctc tgttctgcat 4020 gcttgctgct ttaccctggc actttccttc tccaccatga gtgcgagtcc tgggagtcct 4080 gggagggtga ggattaatgc cagcctgggg agcagatagc tgacagagtc cttgggtaac 4140 tggcttgaac caggacctca ggattccact ctggggatct agctttgtct gggccagtga 4200 agatctctat aatggcatta ttgccagggg ataaacattt cactgggttc tgatctgttg 4260 ggtgtggctt cctggaaaat atggtgagag gaattctgct aaggatacaga ttgataagaa 4320 agttctgaga ttgattagta atgcctgcct tggactcagg aagggaagtg gcagtatgaa 4380 tgccatgtct taatcatttt ggttaaaata tgcttcccaa aagatttcca cgtgtgttct 4440 tgtttatttg acatctgtct ccatatcagt cttgaaagcc tttctgtgtg tatatatatg 4500 atgtttgcgt gtatatatgt ttttgtgtgt gcatatggaa gtcagaaatc actgggtgtc 4560 ttcctccatt cctttgcaat gtatgttttt ttttttttta cgatttattt actatatgaa 4620 tgttttgcct gaatacatgc ataggtgtca cgtacatgcc tgctggaacg cttggaactg 4680 gagttacagg tggctatgag ctacagtgtg agcactggga atcaaacctg ggtcttctgc 4740 aagagcaaca aattaaaagt cagctcttaa ctacttgagc tatttttcca actcc 4795 <210> 2 <211> 309 <212> PRT <213> Mus musculus <400> 2 Met Gly Val Pro Ala Val Pro Glu Ala Ser Ser Pro Arg Trp Gly Thr 1 5 10 15 Leu Leu Leu Ala Ile Phe Leu Ala Ala Ser Arg             20 25 30 Phe Lys Val Thr Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln         35 40 45 Asn Ala Thr Leu Thr Cys Arg Ile Leu Gly Pro Val Ser Lys Gly His     50 55 60 Asp Val Thr Ile Tyr Lys Thr Trp Tyr Leu Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Met Cys Lys Glu His Arg Pro Ile Arg Asn Phe Thr Leu Gln His                 85 90 95 Leu Gln His His Gly Ser His Leu Lys Ala Asn Ala Ser His Asp Gln             100 105 110 Pro Gln Lys His Gly Leu Glu Leu Ala Ser Asp His His Gly Asn Phe         115 120 125 Ser Ile Thr Leu Arg Asn Val Thr Pro Arg Asp Ser Gly Leu Tyr Cys     130 135 140 Cys Leu Val Ile Glu Leu Lys Asn His His Pro Glu Gln Arg Phe Tyr 145 150 155 160 Gly Ser Met Glu Leu Gln Val Gln Ala Gly Lys Gly Ser Gly Ser Ths                 165 170 175 Cys Met Ala Ser Asn Glu Gln Asp Ser Asp Ser Ile Thr Ala Ala Ala             180 185 190 Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu Pro Leu Ile         195 200 205 Leu Leu Leu Val Tyr Lys Gln Arg Gln Val Ala Ser His Arg Arg Ala     210 215 220 Gln Glu Leu Val Arg Met Asp Ser Ser Asn Thr Gln Gly Ile Glu Asn 225 230 235 240 Pro Gly Phe Glu Thr Pro Pro Phe Gln Gly Met Pro Glu Ala Lys                 245 250 255 Thr Arg Pro Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser Glu Ser             260 265 270 Gly Arg Tyr Leu Leu Ser Asp Pro Ser Thr Pro Leu Ser Pro Pro Gly         275 280 285 Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Val Pro Asp Ser Pro     290 295 300 Asn Ser Glu Ala Ile 305 <210> 3 <211> 4774 <212> DNA <213> Homo sapiens <400> 3 gggggcgggt gcctggagca cggcgctggg gccgcccgca gcgctcactc gctcgcactc 60 agtcgcggga ggcttccccg cgccggccgc gtcccgcccg ctccccggca ccagaagttc 120 ctctgcgcgt ccgacggcga catgggcgtc cccacggccc tggaggccgg cagctggcgc 180 tggggatccc tgctcttcgc tctcttcctg gctgcgtccc taggtccggt ggcagccttc 240 aaggtcgcca cgccgtattc cctgtatgtc tgtcccgagg ggcagaacgt caccctcacc 300 tgcaggctct tgggccctgt ggacaaaggg cacgatgtga ccttctacaa gacgtggtac 360 cgcagctcga ggggcgaggt gcagacctgc tcagagcgcc ggcccatccg caacctcacg 420 ttccaggacc ttcacctgca ccatggaggc caccaggctg ccaacaccag ccacgacctg 480 gctcagcgcc acgggctgga gtcggcctcc gaccaccatg gcaacttctc catcaccatg 540 cgcaacctga ccctgctgga tagcggcctc tactgctgcc tggtggtgga gatcaggcac 600 caccactcgg agcacagggt ccatggtgcc atgagctgtca ggtgcagac aggcaaagat 660 gcaccatcca actgtgtggt gtacccatcc tcctcccagg atagtgaaaa catcacggct 720 gcagccctgg ctacgggtgc ctgcatcgta ggaatcctct gcctccccct catcctgctc 780 ctggtctaca agcaaaggca ggcagcctcc aaccgccgtg cccaggagct ggtgcggatg 840 gacagcaaca ttcaagggat tgaaaacccc ggctttgaag cctcaccacc tgcccagggg 900 atacccgagg ccaaagtcag gcaccccctg tcctatgtgg cccagcggca gccttctgag 960 tctgggcggc atctgctttc ggagcccagc acccccctgt ctcctccagg ccccggagac 1020 gtcttcttcc catccctgga ccctgtccct gactctccaa actttgaggt catctagccc 1080 agctggggga cagtgggctg ttgtggctgg gtctggggca ggtgcatttg agccagggct 1140 ggctctgtga gtggcctcct tggcctcggc cctggttccc tccctcctgc tctgggctca 1200 gatactgtga catcccagaa gcccagcccc tcaacccctc tggatgctac atggggatgc 1260 tggacggctc agcccctgtt ccaaggattt tggggtgctg agattctccc ctagagacct 1320 gaaattcacc agctacagat gccaaatgac ttacatctta agaagtctca gaacgtccag 1380 cccttcagca gctctcgttc tgagacatga gccttgggat gtggcagcat cagtgggaca 1440 agatggacac tgggccaccc tcccaggcac cagacacagg gcacggtgga gagacttctc 1500 ccccgtggcc gccttggctc ccccgttttg cccgaggctg ctcttctgtc agacttcctc 1560 tttgtaccac agtggctctg gggccaggcc tgcctgccca ctggccatcg ccaccttccc 1620 cagctgcctc ctaccagcag tttctctgaa gatctgtcaa caggttaagt caatctgggg 1680 cttccactgc ctgcattcca gtccccagag cttggtggtc ccgaaacggg aagtacatat 1740 tggggcatgg tggcctccgt gagcaaatgg tgtcttgggc aatctgaggc caggacagat 1800 gttgccccac ccactggaga tggtgctgag ggaggtgggt ggggccttct gggaaggtga 1860 gtggagaggg gcacctgccc cccgccctcc ccatccccta ctcccactgc tcagcgcggg 1920 ccattgcaag ggtgccacac aatgtcttgt ccaccctggg acacttctga gtatgaagcg 1980 ggatgctatt aaaaactaca tggggaaaca ggtgcaaacc ctggagatgg attgtaagag 2040 ccagtttaaa tctgcactct gctgctcctc ccccaccccc accttccact ccatacaatc 2100 tgggcctggt ggagtcttcg cttcagagcc attcggccag gtgcgggtga tgttcccatc 2160 tcctgcttgt gggcatgccc tggctttgtt tttatacaca taggcaaggt gagtcctctg 2220 tggaattgtg attgaaggat tttaaagcag gggaggagag tagggggcat ctctgtacac 2280 tctgggggta aaacagggaa ggcagtgcct gagcatgggg acaggtgagg tggggctggg 2340 cagaccccct gtagcgttta gcaggatggg ggccccaggt actgtggaga gcatagtcca 2400 gcctgggcat ttgtctccta gcagcctaca ctggctctgc tgagctgggc ctgggtgctg 2460 aaagccagga tttggggcta ggcgggaaga tgttcgccca attgcttggg gggttggggg 2520 gatggaaaag gggagcacct ctaggctgcc tggcagcagt gagccctggg cctgtggcta 2580 cagccaggga accccacctg gacacatggc cctgcttcta agccccccag ttaggcccaa 2640 aggaatggtc cactgagggc ctcctgctct gcctgggctg ggccaggggc tttgaggaga 2700 gggtaaacat aggcccggag atggggctga cacctcgagt ggccagaata tgcccaaacc 2760 ccggcttctc ccttgtccct aggcagaggg gggtcccttc ttttgttccc tctggtcacc 2820 acaatgcttg atgccagctg ccataggaag agggtgctgg ctggccatgg tggcacacac 2880 ctgtcctccc agcactttgc agggctgagg tggaaggacc gcttaagccc aggtgttcaa 2940 ggctgctgtg agctgtgttc gagccactac actccagcct ggggacggag caaaactttg 3000 cctcaaaaca aattttaaaa agaaagaaag aaggaaagag ggtatgtttt tcacaattca 3060 tgggggcctg catggcagga gtggggacag gacacctgct gttcctggag tcgaaggaca 3120 agcccacagc ccagattccg gttctcccaa ctcaggaaga gcatgccctg ccctctgggg 3180 aggctggcct ggccccagcc ctcagctgct gaccttgagg cagagacaac ttctaagaat 3240 ttggctgcca gaccccaggc ctggctgctg ctgtgtggag agggaggcgg cccgcagcag 3300 aacagccacc gcacttcctc ctcagcttcc tctggtgcgg ccctgccctc tcttctctgg 3360 acccttttac aactgaacgc atctgggctt cgtggtttcc tgttttcagc gaaatttact 3420 ctgagctccc agttccatct tcatccatgg ccacaggccc tgcctacaac gcactaggga 3480 cgtccctccc tgctgctgct ggggaggggc aggctgctgg agccgccctc tgagttgccc 3540 tgggtgcgag aacatggcgc ctccaggggg cgggaggagc actaggggct ggggagag 3660 gctcctggag cgctggattc ctggcacagt ctgaggccct gagagggaaa tccatgcttt 3720 taagaactaa ttcattgtta ggagatcaat caggaattag gggccatctt acctatctcc 3780 tgacattcac agtttaatag agacttcctg cctttattcc ctcccaggga gaggctgaag 3840 gaatggaatt gaaagcacca tttggagggt tttgctgaca cagcggggac tgctcagcac 3900 tccctaaaaa cacaccatgg aggccactgg tgactgctgg tgggcaggct ggccctgcct 3960 gggggagtcc gtggcgatgg gcgctggggt ggaggtgcag gagccccagg acctgctttt 4020 caaaagactt ctgcctgacc agagctccca ctacatgcag tggcccaggg cagaggggct 4080 gatacatggc ctttttcagg gggtgctcct cgcggggtgg acttgggagt gtgcagtggg 4140 acagggggct gcaggggtcc tgccaccacc gagcaccaac ttggcccctg gggtcctgcc 4200 tcatgaatga ggccttcccc agggctggcc tgactgtgct gggggctggg ttaacgtttt 4260 ctcagggaac cacaatgcac gaaagaggaa ctggggttgc taaccaggat gctgggaaca 4320 aaggcctctt gaagcccagc cacagcccag ctgagcatga ggcccagccc atagacggca 4380 caggccacct ggcccattcc ctgggcattc cctgctttgc attgctgctt ctcttcaccc 4440 catggaggct atgtcaccct aactatcctg gaatgtgttg agagggattc tgaatgatca 4500 atatagcttg gtgagacagt gccgagatag atagccatgt ctgccttggg cacgggagag 4560 ggaagtggca gcatgcatgc tgtttcttgg ccttttctgt tagaatactt ggtgctttcc 4620 aacacacttt cacatgtgtt gtaacttgtt tgatccaccc ccttccctga aaatcctggg 4680 aggttttatt gctgccattt aacacagagg gcaatagagg ttctgaaagg tctgtgtctt 4740 gtcaaaacaa gtaaacggtg gaactacgac taaa 4774 <210> 4 <211> 311 <212> PRT <213> Homo sapiens <400> 4 Met Gly Val Pro Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser 1 5 10 15 Leu Leu Phe Ala Leu Phe Leu Ala Ala Ser Leu             20 25 33 Phe Lys Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln         35 40 45 Asn Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His     50 55 60 Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 65 70 75 80 Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp                 85 90 95 Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp             100 105 110 Leu A1a Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn         115 120 125 Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr     130 135 140 Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His Arg Val 145 150 155 160 His Gly Ala Met Glu Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser                 165 170 175 Asn Cys Val Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr             180 185 190 Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu         195 200 205 Pro Leu Il Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn     210 215 220 Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile 225 230 235 240 Glu Asn Pro Gly Phe Glu Ala Ser Pro Ala Gln Gly Ile Pro Glu                 245 250 255 Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser             260 265 270 Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr Pro Leu Ser Pro         275 280 285 Pro Gly Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp     290 295 300 Ser Pro Asn Phe Glu Val Ile 305 310 <210> 5 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence for PD-L3 <400> 5 Ile Thr Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu 1 5 10 15 Cys Leu Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln             20 25 30 <210> 6 <211> 1316 <212> DNA <213> Mus musculus <400> 6 ggagtcctcc ccttggagcc tgggaggcct agggagaaag tagttctctt tcggtggcag 60 ggttgctgtc gagggcaccg agcaggagga taggtcgaca gagacgagga gttctggctc 120 ctcctgcaga catgcaccag cggctgctgg gctcgtccct gggrctcgcc cccgcgcggg 180 ggctctgaat gcctgccgcc gcccccatga gagcaccggc ctgggctccc gcccctaagc 240 ctctgctcgc ggagactgag ccatgtgggc ctggggctgg gccgctgcag cgctcctctg 300 gctacagact gcaggagccg gggcccggca ggagctcaag aagtctcggc agctgtttgc 360 gcgtgtggat tcccccaata ttaccacgtc caaccgtgag ggattcccag gctccgtcaa 420 gcccccggaa gcctctggac ctgagctctc agatgcccac atgacgtggt tgaactttgt 480 ccgacggcca gatgatgggt cctctagaaa acggtgtcgt ggccgggaca agaagtcgcg 540 aggcctctca ggtctcccag ggcccccagg acctcctggc cctcctggtc cccctggctc 600 ccctggtgtg ggcgttaccc cagaggcctt actgcaggaa tttcaggaga tactgaaaga 660 ggccacagaa cttcgattcc cagggctacc agacacattg ttaccccagg aacccagcca 720 acggctggtg gttgaggcct tctactgccg tttgaaaggc cctgtgctgg tggacaagaa 780 gactctggtg gaactgcaag gattccaagc tcctactact cagggcgcct tcctgcgggg 840 atctggcctg agcctgtcct tgggccgatt cacagcccca gtctctgcca tcttccagtt 900 ttctgccagc ctgcacgtgg accacagtga actgcagggc agaggccggt tgcgtacccg 960 ggatatggtc cgtgttctca tctgtattga gtccttgtgt catcgtcata cgtccctgga 1020 ggctgtatca ggtctggaga gcaacagcag ggtcttcaca gtgcaggttc aggggctgct 1080 gcatctacag tctggacagt atgtctctgt gttcgtggac aacagttctg gggcagtcct 1140 caccatccag aacacttcca gcttctcggg aatgcttttg ggtacctagc ggagctgaag 1200 aaacgattgt ggattgagga accaacacct tgcttcttag aggagctgaa aaggactact 1260 cactcccctt ttaatagttt tcatagcaat aaagaactcc aaacttcttc atcgct 1316 <210> 7 <211> 308 <212> PRT <213> Mus musculus <400> 7 Met Trp Ala Trp Gly Trp Ala Ala Ala Leu Leu Trp Leu Gln Thr 1 5 10 15 Ala Gly Ala Gly Ala Arg Gln Glu Leu Lys Lys Ser Arg Gln Leu Phe             20 25 30 Ala Arg Val Asp Ser Pro Asn Ile Thr Thr Ser Asn Arg Glu Gly Phe         35 40 45 Pro Gly Ser Val Lys Pro Pro Glu Ala Ser Gly Pro Glu Leu Ser Asp     50 55 60 Ala His Met Thr Trp Leu Asn Phe Val Arg Arg Pro Asp Asp Gly Ser 65 70 75 80 Ser Arg Lys Arg Cys Arg Gly Arg Asp Lys Lys Ser Arg Gly Leu Ser                 85 90 95 Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly             100 105 110 Ser Pro Gly Val Gly Val Thr Pro Glu Ala Leu Leu Gln Glu Phe Gln         115 120 125 Glu Ile Leu Lys Glu Ala Thr Glu Leu Arg Phe Ser Gly Leu Pro Asp     130 135 140 Thr Leu Leu Pro Gln Glu Pro Ser Gln Arg Leu Val Val Glu Ala Phe 145 150 155 160 Tyr Cys Arg Leu Lys Gly Pro Val Leu Val Asp Lys Lys Thr Leu Val                 165 170 175 Glu Leu Gln Gly Phe Gln Ala Pro Thr Gln Gly Ala Phe Leu Arg             180 185 190 Gly Ser Gly Leu Ser Leu Ser Leu Gly Arg Phe Thr Ala Pro Val Ser         195 200 205 Ala Ile Phe Gln Phe Ser Ala Ser Leu His Val Asp His Ser Glu Leu     210 215 220 Gln Gly Arg Gly Arg Leu Arg Thr Arg Asp Met Val Arg Val Leu Ile 225 230 235 240 Cys Ile Glu Ser Leu Cys His Arg His Thr Ser Leu Glu Ala Val Ser                 245 250 255 Gly Leu Glu Ser Asn Ser Arg Val Phe Thr Val Gln Val Gln Gly Leu             260 265 270 Leu His Leu Gln Ser Gly Gln Tyr Val Ser Val Phe Val Asp Asn Ser         275 280 285 Ser Gly Ala Val Leu Thr Ile Gln Asn Thr Ser Ser Phe Ser Gly Met     290 295 300 Leu Leu Gly Thr 305 <210> 8 <211> 1055 <212> DNA <213> Homo sapiens <400> 8 ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg 60 tggtccccct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac 120 agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc agcgcgtcct 180 cccgcgaggg gctgcccgag gcccccaagc catcccaggc ctcaggacct gagttctccg 240 acgcccacat gacatggctg aactttgtcc ggcggccgga cgacggcgcc ttaaggaagc 300 ggtgcggaag cagggacaag aagccgcggg atctcttcgg tcccccagga cctccaggtg 360 cagaagtgac cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg 420 agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc ctgcggctgg 480 tgggcgaggc ctttcactgc cggctgcagg gtccccgccg ggtggacaag cggacgctgg 540 tggagctgca tggtttccag gctcctgctg cccaaggtgc cttcctgcga ggctccggtc 600 tgagcctggc ctcgggtcgg ttcacggccc ccgtgtccgg catcttccag ttctctgcca 660 gtctgcacgt ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg 720 tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg gaggccgtct 780 caggcctgga gagcaacagc agggtcttca cgctacaggt gcaggggctg ctgcagctgc 840 aggctggaca gtacgcttct gtgtttgtgg acaatggctc cggggccgtc ctcaccatcc 900 aggcgggctc cagcttctcc gggctgctcc tgggcacgtg agggcgccca ggggggctgg 960 cgaggagctg ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa 1020 agagccctcc accctcaaaa aaaaaaaaaa aaaaa 1055 <210> 9 <211> 302 <212> PRT <213> Homo sapiens <400> 9 Met Arg Arg Trp Ala Trp Ala Ala Val Val Leu Leu Gly Pro Gln 1 5 10 15 Leu Val Leu Leu Gly Gly Val Gly Ala Arg Arg Glu Ala Gln Arg Thr             20 25 30 Gln Gln Pro Gly Gln Arg Ala Asp Pro Pro Asn Ala Thr Ala Ser Ala         35 40 45 Ser Ser Arg Glu Gly Leu Pro Glu Ala Pro Lys Pro Ser Gln Ala Ser     50 55 60 Gly Pro Glu Phe Ser Asp Ala His Met Thr Trp Leu Asn Phe Val Arg 65 70 75 80 Arg Pro Asp Asp Gly Ala Leu Arg Lys Arg Cys Gly Ser Arg Asp Lys                 85 90 95 Lys Pro Arg Asp Leu Phe Gly Pro Pro Gly Pro Pro Gly Ala Glu Val             100 105 110 Thr Ala Glu Thr Leu Leu His Glu Phe Gln Glu Leu Leu Lys Glu Ala         115 120 125 Thr Glu Arg Arg Phe Ser Gly Leu Leu Asp Pro Leu Leu Pro Gln Gly     130 135 140 Ala Gly Leu Arg Leu Val Gly Glu Ala Phe His Cys Arg Leu Gln Gly 145 150 155 160 Pro Arg Arg Val Asp Lys Arg Thr Leu Val Glu Leu His Gly Phe Gln                 165 170 175 Ala Pro Ala Ala Gln Gly Ala Phe Leu Arg Gly Ser Gly Leu Ser Leu             180 185 190 Ala Ser Gly Arg Phe Thr Ala Pro Val Ser Gly Ile Phe Gln Phe Ser         195 200 205 Ala Ser Leu His Val Asp His Ser Glu Leu Gln Gly Lys Ala Arg Leu     210 215 220 Arg Ala Arg Asp Val Val Cys Val Leu Ile Cys Ile Glu Ser Leu Cys 225 230 235 240 Gln Arg His Thr Cys Leu Glu Ala Val Ser Gly Leu Glu Ser Asn Ser                 245 250 255 Arg Val Phe Thr Leu Gln Val Gln Gly Leu Leu Gln Leu Gln Ala Gly             260 265 270 Gln Tyr Ala Ser Val Phe Val Asp Asn Gly Ser Gly Ala Val Leu Thr         275 280 285 Ile Gln Ala Gly Ser Ser Phe Ser Gly Leu Leu Leu Gly Thr     290 295 300 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence for Treg-sTNF <400> 10 Ile Phe Gln Phe Ser Ala Ser Leu His Val Asp His Ser Glu Leu Gln 1 5 10 15 Gly

Claims (54)

A pharmaceutical composition for inducing immunosuppression or treating an allergic, inflammatory or autoimmune disease in an individual in need of immunosuppression or treatment, comprising a multimeric VISTA protein, isolated or recombinant immunosuppressive,
The multimer VISTA protein
Wherein the polypeptide comprises from 2 to 6 copies of a polypeptide consisting of the extracellular domain of a human or murine VISTA polypeptide of SEQ ID NO: 2 or 4. The pharmaceutical composition of claim 1, wherein at least one of the extracellular domains is attached to the N-terminus of the oligomerization domain. 3. The method of claim 2 wherein the oligomerization domain is selected from GCN4, COMP, SNARE, CMP, MAT, LLR, 1 NLRC, NOD2 nucleotide-binding NLRC2, 1 NLRC NOD2 nucleotide-binding NLRC2 with LRR, Gt; 4. The pharmaceutical composition according to any one of claims 1 to 3, wherein at least one of the extracellular domains is attached to the Ig polypeptide directly or indirectly. 4. The method according to any one of claims 1 to 3, wherein at least one other immunosuppressant selected from PD-1, PD-L1, PD-L2, CTLA4 and ICOS proteins and any of the at least one other immunosuppressant Lt; / RTI &gt; antibody. 4. The method according to any one of claims 1 to 3, wherein the allergic, inflammatory or autoimmune disease is psoriasis, dermatitis, atopic dermatitis; Systemic scleroderma, sclerosis; Crohn's disease, ulcerative colitis; Respiratory distress syndrome, adult respiratory distress syndrome (ARDS); dermatitis; Meningitis; Encephalitis; Uveitis; Colitis; Glomerulonephritis; Eczema, asthma, atherosclerosis; Leukocyte adhesion deficiency; Rheumatoid arthritis; Systemic lupus erythematosus (SLE); Diabetes mellitus; Multiple sclerosis; Reynaud's syndrome; Autoimmune thyroiditis; Allergic encephalomyelitis; Sjorgen's syndrome; Juvenile onset diabetes; Tuberculosis, sarcoidosis, polymyositis, granulomatosis, vasculitis; Pernicious anemia (Addison's disease); Central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome; Hemolytic anemia, cryoglobinemia or Coombs positive anemia; Myasthenia gravis; Antigen-antibody complex mediated diseases; Anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; Pemphigoid bullous; Pemphigus; Autoimmune polyendocrinopathies; Reiter's disease; Stiff-man syndrome; Behcet disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM polyneuropathies; (ITP), autoimmune thrombocytopenia, arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis Inflammatory arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spinal arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, The present invention relates to a method of treating psoriasis, including psoriasis, psoriasis, plaque psoriasis, guttae psoriasis, psoriatic arthritis, psoriasis, psoriatic arthritis, psoriatic arthritis, psoriatic arthritis, psoriatic arthritis, polyarthritis chronica primaria, reactive arthritis, ankylosing spondylitis, pustular psoriasis, psoriasis of the nails, dermatitis, contact dermatitis, Allergic contact dermatitis, dermatitis herpetiformis, atopic dermatitis, X-linked and IgM syndrome, urticaria, chronic allergic urticaria, chronic idiopathic urticaria, , Chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic sclerosis, sclerosis, systemic sclerosis, Multiple sclerosis (MS), spino-optical MS (MS), primary progressive MS (MS) PPMS, relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, ulcerative colitis, ulcerative colitis, Inflammatory bowel disease, colitis polyposa, neonatal necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, Primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (adu or acute respiratory distress syndrome (ARDS), meningitis, all or part of the uveal inflammation, iritis, choroiditis, autoimmune hematological disorder, rheumatoid spondylitis rheumatoid spondylitis), sudden hearing loss, IgE-mediated disease, anaphylaxis, allergic rhinitis, atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic encephalitis, brainstem encephalitis ), Uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, autoimmune uveitis), glomerulonephritis (glomerulonephritis; GN, idiopathic membranous GN, or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN), rapid progressive GN, allergic conditions, autoimmunity Myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), systemic lupus erythematosus, skin SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome neuritis, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus, nephritis, cerebritis, pediatric lupus, non-renal lupus, Diabetic retinopathy, extra-renal lupus, discoid lupus, alopecia, juvenile onset type I diabetes mellitus, pediatric insulin-dependent diabetes mellitus, Immune responses related to acute and delayed hypersensitivity mediated by abetes mellitus (IDDM), adult diabetes (type II diabetes), autoimmune diabetes mellitus, idiopathic diabetes insipidus, cytokines and T lymphocytes, tuberculosis, sarcoidosis The present invention relates to a method for the treatment of sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, vasculitis, vasculitis, rheumatic polymyalgia rheumatica, giant cell (Takayasu) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microalbuminuria microscopic polyarteritis, CNS vasculitis, necrotising, dermal, or hypersensitivity vasculitis, systemic necrotizing vasculitis itis), ANCA-associated vasculitis, Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive Anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia, autoimmune hemolytic anemia, AIDS), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, CNS inflammatory disorders, multiorgan injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody, leukopenia, leukopenia, leukopenia, leukocyte diapedesis Syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, Alzheimer ' s syndrome, Alzheimer ' s syndrome, Alzheimer ' Stevens-Johnson syndrome, pemphigoid, pemphigoid bullous, skin pemphigoid, pemphigus, pemphigoid, Pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease, ) Or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathy, IgM-mediated neuropathy, thrombocytopenia ), Thrombotic thrombocytopenic purpura (Thrombotic thrombocytopenic purpura; TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune testitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis, ); Idiopathic hypothyroidism, Grave's disease, polyglandular syndrome, autoimmune polyglandular syndromes, polyglandular endocrinopathy syndromes (GI) syndromes, autoimmune thyroid disease, idiopathic hypothyroidism, Paraneoplastic syndromes, neurologic paraneoplastic syndromes, Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, Stiff-Man syndrome, stiff-man syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-related Thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, , Opsoclonus or ossoclonus myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, (Eg, chronic hepatitis, chronic hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, Bronchiolitis obliterans versus NSIP, Guillain-Barre syndrome, Berger's disease, IgA nephropathy, idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, , Pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Celiac disease, Celiac disease, Fruit (celiac sprue) (Chapter gluten neuropathy (gluten enteropathy)), refractory sprue (refractory sprue), idiopathic sprue (idiopathic sprue), cold globulin hyperlipidemia (cryoglobulinemia), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis; ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease, autoimmune inner ear disease (AGED), autoimmune hearing loss, (OCD), polychondritis, refractory or recurrent polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, noncancerous lymphocytic leukemia, The present invention relates to a method for the treatment and prophylaxis of non-cancerous lymphocytosis, primary lymphocytosis, monoclonal B cell lymphopenia, peripheral neuropathy, paraneoplastic syndrome, channelopathies, epilepsy, (Eg, migraine, arrhythmia, muscle disorders, deafness, blindness, periodic paralysis, CNS channelopathy, autism, inflammatory mastitis focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, ), Multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases, autoimmune dehydration, Diabetic nephropathy, Dressler ' s syndrome, alopecia areata, CREST syndrome, calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma sclerodactyl, telangiectasia, male and female autoimmune sterility, mixed connective tissue diseases, Chagas' disease, rheumatism ic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird fever (bird -fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis, allergic alveolitis, fibrotic alveolitis, epilepsy The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment and prophylaxis of an infectious disease, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, Aspergillosis, Sampter ' s syndrome, Caplan ' s syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fi bradycardia, bradycardia, brosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis, chronic cystitis, dysplastic cystitis, Iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) HIV infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, Rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, streptococcal infection, The present invention relates to a method of treating a condition selected from the group consisting of poststreptococcal nephritis, obstructive thrombotic inflammation, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, inflammatory keratoconjunctivitis, idiopathic nephritic syndrome, , Minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, Inflammatory bowel disease, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis, autoimmune hemolysis, Dopuytren's contracture, endophthalmia phacoanaphylactica, allergic enteritis, erythema nodosum leprosum, erythema nodosum leprosum, erythema nodosum leprosum, ), Idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich s disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, infectious mononucleosis (mononucleosis infectiosa) ), Transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, acute multiple neuromyositis pyoderma gangrenosum, Quervain's thyreoiditis, acquired spleen atrophy, infertility due to antifungal antibodies, non-malignant thymoma, vitiligo, SCID, Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome, AIDS), parasitic disease, Leschmania, toxic-shock syndrome, food poisoning, conditions associated with T cell infiltration, leukocyte-adhesion deficiency, cytokines and T lymphocytes An organ-antibody complex-mediated disease, an antiglomerular basement membrane disease, an allergic neuritis, an autoimmune disease, an autoimmune disease, an autoimmune disease associated with acute and delayed hypersensitivity, a disease associated with leukocyte leukemia, multiple organ injury syndrome, allergic neuritis, autoimmune polyendocrinopathies, ovarian inflammation, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, Mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy (periph erosive neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, , Epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, eosinophilia-myalgia syndrome, eosinophilia-myalgia syndrome, eosinophilia, eosinophilia, eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler ' s syndrome, chronic eosinophilic pneumonia, tropical pulmonary &lt; RTI ID = 0.0 &gt; eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, multiple myeloproliferative disorders, eosinophilia, bronchopneumonic aspergillosis, Inflammatory bowel disease, endocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, Transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, neurological diseases, ischemic reperfusion Impairment of blood pressure response, vascular dysfunction, vasodilation, tissue damage, cardiovascular ischemia atherosclerosis, emia, hyperalgesia, cerebral ischemia, diseases accompanied by vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion of cardiac muscle or other tissues Dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory diseases, ocular and orbital inflammatory diseases, granulocyte transfusion-associated syndromes, cytokine-induced Toxic, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disease, arterial disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis. s). &lt; / RTI &gt; The pharmaceutical composition according to claim 6, wherein the allergic, inflammatory or autoimmune disease is multiple sclerosis or rheumatoid arthritis. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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